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, and
Centre for Human Nutrition, Department of Biochemistry, The University of Western Ontario, London, Ontario N6A 5C1, Canada; * Palm Oil Research Institute of Malaysia, 50720, Kuala Lumpur, Malaysia; and Department of Oncology, The University of Western Ontario, London Regional Cancer Centre, London, Ontario N6A 4L6, Canada
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGMENTS
LITERATURE CITED
ABSTRACT 
Tocotrienols are a form of vitamin E, having an unsaturated isoprenoid side-chain rather than the saturated side-chain of tocopherols. The tocotrienol-rich fraction (TRF) from palm oil contains 
-tocopherol and a mixture of -, 
- and 
-tocotrienols. Earlier studies have shown that tocotrienols display anticancer activity. We previously reported that TRF, -, - and -tocotrienols inhibited proliferation of estrogen receptor-negative MDA-MB-435 human breast cancer cells with 50% inhibitory concentrations (IC50) of 180, 90, 30 and 90 µg/mL, respectively, whereas -tocopherol had no effect at concentrations up to 500 µg/mL. Further experiments with estrogen receptor-positive MCF-7 cells showed that tocotrienols also inhibited their proliferation, as measured by [3H] thymidine incorporation. The IC50s for TRF, -tocopherol, -, - and -tocotrienols were 4, 125, 6, 2 and 2 µg/mL, respectively. Tamoxifen, a widely used synthetic antiestrogen inhibits the growth of MCF-7 cells with an IC50 of 0.04 µg/mL. We tested 1:1 combinations of TRF, -tocopherol and the individual tocotrienols with tamoxifen in both cell lines. In the MDA-MB-435 cells, all of the combinations were found to be synergistic. In the MCF-7 cells, only 1:1 combinations of - or -tocotrienol with tamoxifen showed a synergistic inhibitory effect on the proliferative rate and growth of the cells. The inhibition by tocotrienols was not overcome by addition of excess estradiol to the medium. These results suggest that tocotrienols are effective inhibitors of both estrogen receptor-negative and -positive cells and that combinations with tamoxifen should be considered as a possible improvement in breast cancer therapy.
INTRODUCTION
Experiments in other laboratories have shown that diets containing a high level of palm oil do not promote mammary carcinogenesis in rats (Kritchevsky et al. 1992
, Sundram et al. 1989). Saturated fats, such as coconut oil and beef tallow, also failed to promote mammary carcinogenesis in rats (Carroll and Khor 1971) evidently because they contain insufficient polyunsaturated fatty acid (linoleic acid) (Ip 1987). However, this does not appear to be the limiting factor with palm oil.
Palm oil stripped of its vitamin E fraction promoted mammary carcinogenesis like other unsaturated fats and oils, and addition of the vitamin E fraction from palm oil to a high corn oil diet tended to counteract the promoting effect of the dietary corn oil (Nesaretnum et al. 1992). The vitamin E of palm oil, unlike that of most other fats and oils, consists largely of tocotrienols, the remainder being -tocopherol (Fig. 1) (Ong 1993, Sundram et al. 1989). This fraction of the oil, referred to as the tocotrienol-rich fraction (TRF)4 or Palm Vitee, was shown to inhibit the proliferation and growth of human breast cancer cells in vitro, whereas -tocopherol was ineffective. The individual tocotrienols of TRF were also found to inhibit the cancer cells more effectively than would be expected from results obtained with the TRF itself (Carroll et al. 1995).
The above experiments were conducted with estrogen receptor-negative MDA-MB-435 cancer cells (Price et al. 1990). The ability of TRF and tocotrienols to inhibit MCF-7 estrogen receptor-positive cells (Soule et al. 1973) was subsequently investigated. TRF and the individual tocotrienols inhibited this line more effectively than -tocopherol, but not as effectively as tamoxifen (Guthrie et al. 1995).
Tamoxifen, an estrogen antagonist, is used extensively in the hormonal therapy of breast cancer (Jordan 1994, Powles and Hickish 1995). Tamoxifen acts mainly by blocking the stimulatory action of estrogens in hormone-responsive breast cancer cells (Haran et al. 1994, Jordan 1995), but has dual dose-dependent estrogenic/antiestrogenic properties (Horwitz et al. 1978). The use of tamoxifen is limited, however, by the development of resistance to this compound in most patients (Osborne et al. 1995). Because many recurrent breast cancers are estrogen receptor-independent, a drug or dietary agent that inhibits the growth of both estrogen receptor-positive and -negative tumors would be of great interest (Peterson and Barnes 1991).
In this paper, we report the effects of TRF, -tocopherol and the individual tocotrienols of palm oil on MCF-7 estrogen-responsive human breast cancer cells. The compounds were also tested in 1:1 combinations with tamoxifen on both MCF-7 and MDA-MB-435 cells.
MATERIALS AND METHODS
-tocopherol and the individual tocotrienols were obtained from the Palm Oil Research Institute of Malaysia (PORIM), Kuala Lumpur. Tamoxifen and 17-
estradiol were purchased from Sigma Chemical (St. Louis, MO). MDA-MB-435 cells (Price et al. 1990) were obtained from Janet Price (M. D. Anderson Cancer Center, Houston, TX) and MCF-7 (Soule et al. 1973) cells were obtained from the American Tissue Culture Collection (Rockville, MD). Tissue culture medium and fetal calf serum were purchased from GIBCO (Burlington, ON). Fetal calf serum treated with dextran-coated charcoal (FCS/DCC) was purchased from Cocalico Biologicals (Reamstown, PA). Trypsin was obtained from Difco Laboratories (Detroit, MI) and [3H] thymidine (248 Bg/mmol) was purchased from ICN (Irvine, CA). All other chemicals were from Sigma.
-tocopherol and tocotrienols.
Cell culture. MDA-MB-435 cells (estrogen receptor-negative human breast cancer cells) were maintained at 37°C in minimum essential medium containing 3.7g of NaHCO3/L, supplemented with 10% (v/v) FCS. The medium was equilibrated with a humidified atmosphere of 5% CO2. Stock cultures were seeded at a density of 2 × 105 cells/mL and allowed to multiply for 48-72 h.
-tocopherol, -, -, and -tocotrienols were dissolved in dimethyl sulfoxide (DMSO) at a concentration of 50 mg/mL and then diluted into the culture medium so that the final concentration of DMSO was <0.1%. The same amount of DMSO was added to control cells. Tamoxifen was dissolved in ethanol at a concentration of 50 mg/mL and a similar control was conducted.
Incorporation of [3H] thymidine into DNA.
MDA-MB-435 cells were plated at 2 × 104 cells/well in 96-well, flat-bottomed culture plates in a total volume of 200 µL of medium and incubated at 37°C for 48 h with or without test compounds. [3H] Thymidine (18.5 µBg/well) was then added; after 4 h, the cells were harvested onto a glass fiber filter paper using a semiautomatic 12-well cell harvester (Skatron, Sterling, VA). Radioactivity on the filter paper was counted, using Scintiverse in a liquid scintillation counter (Kothapalli et al. 1993). For the MCF-7 cells, 5 d prior to use, the growth medium was exchanged for phenol red-free medium containing 10% FCS that had been treated with dextran-coated charcoal (DCC). The cells were then trypsinized and 2 × 104 cells/well were plated as described above. Two days later, the medium was replaced with an experimental one containing 2.5% FCS/DCC and the test compounds for 5 d (Thomas and Monet 1992). Untreated cells were used as a control. [3H] Thymidine was then added and the cells harvested as described above.
Growth experiment.
MDA-MB-435 and MCF-7 cells were plated at 1 × 104 and 5 × 104 cells/dish, respectively, in 60-mm dishes with or without test compounds at their 50% inhibitory concentration (IC50). The cells were removed by trypsinization at the specified times and counted, using a hemacytometer.
Viability of cells.
Viability of cells was measured by an assay described by Hansen et al. (1989). In this assay, a tetrazolium salt, 3-[4,5-dimethylthiazole]-2,5-diphenyltetrazolium bromide (MTT), is converted to a blue formazan product by dehydrogenases that are active in living cells. The intensity of the blue color that develops is a measure of cell viability. MDA-MB-435 and MCF-7 cells (8 × 104) were seeded with various concentrations of the tocotrienols and/or tamoxifen in a 96-well flat-bottomed plate in a total volume of 200 µL of medium. MTT (25 µL of 5 mg/mL) was added to each well. After 3 h, 100 µL of extraction buffer, consisting of 20% SDS, dissolved in a 50% DMF:50% water solution at pH 4.0, was added. The blue color formed was measured at 590 nm.
RESULTS
-tocopherol, tocotrienols and tamoxifen required to inhibit MDA-MB-435 cell proliferation by 50% was determined (Table 1). -Tocotrienol was a more effective inhibitor of these cells than tamoxifen. When TRF, -tocopherol, -, - or -tocotrienols were combined in equimolar concentration with tamoxifen, the combinations inhibited cell proliferative rate much more effectively than the compounds alone (Table 1). Figure 2 shows the inhibition of proliferation and the cytotoxic effect of a 1:1 combination of -tocotrienol and tamoxifen on MDA-MB-435 cells. As can be seen, most cells were viable at the IC50 concentration, suggesting that the compounds are not cytotoxic. Similar results were obtained with all of the compounds and combinations tested. A synergistic effect was also observed when the combinations were tested on growth of these cells over a longer time period, as illustrated in Figure 3.
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Table 1. Inhibition of proliferation MDA-MB-435 cells by TRF and its components, with and without tamoxifen1 |
-tocotrienol and tamoxifen on the proliferation (
) and viability (
) of MDA-MB-435 cells. The cells were incubated with various concentrations of -tocotrienol and tamoxifen for 48 h; [3H] thymidine (18.5 µBg/well) was then added and the cells were harvested after 4 h to evaluate the incorporation of thymidine into DNA. For viability, cells were incubated with various concentrations of -tocotrienol and tamoxifen for 48 h, 3-[4,5-dimethylthiazole]-2,5-diphenyltetrazolium bromide (MTT) was added (25 µL); after 3h, extraction buffer was added (100 µL) and optical density (OD) measurements made at 570 nm. Points are the average of mean values from three experiments.
) or the absence () of 2 µg/mL of -tocotrienol and tamoxifen. Cells were plated in triplicate in 60-mm culture dishes. Cells were removed by trypsinization at the specified times and were counted using a hemacytometer. Each point represents triplicate values.
Effect of TRF,
-tocopherol and the individual tocotrienols with or without tamoxifen on estrogen receptor-positive human breast cancer cells.
TRF and the tocotrienols inhibited the proliferation of MCF-7 cells more effectively than -tocopherol, but not as effectively as tamoxifen (Table 2). The tocotrienols gave much lower IC50s in MCF-7 cells than in MDA-MB-435 cells; - and -tocotrienols were the most potent. When these compounds were tested in 1:1 combination with tamoxifen, the IC50s were in most cases intermediate to those of the individual compounds (Table 2). Only - and -tocotrienols in combination with tamoxifen gave lower IC50s than the compounds alone. The effects of -tocotrienol and tamoxifen, the most inhibitory combination, on cell proliferation and on viability, are illustrated in Figure 4. Most cells were viable at the IC50 concentration. The effect of this combination on growth inhibition is shown in Figure 5.
|
Table 2. Inhibition of proliferation MCF-7 cells by TRF and its components with and without tamoxifen1 |
-tocotrienol and tamoxifen on the proliferation (
) and viability (
) of MCF-7 cells. The cells were incubated for 5 d with various concentrations of -tocotrienol and tamoxifen in phenol red-free medium containing 2.5% fetal calf serum stripped of endogenous steroids. [3H] Thymidine (18.5 µBg/well) was then added; the cells were harvested after 4 h and the incorporation into DNA was measured. For viability, the cells were incubated as for the proliferation assay for 5 d.3-[4,5-Dimethylthiazole]-2,5-diphenyltetrazolium bromide (MTT) was then added (25 µL); after 3 h, extraction buffer was added (100 µL) and optical density (OD) measurements were made at 570 nm. Points are the average of mean values from three experiments.
) or absence (
) of 0.003 µg/mL of 1:1 combinations of -tocotrienol and tamoxifen. Cells were plated in triplicate in 60-mm culture dishes, removed by trypsization at the specified times and counted using a hemocytometer. Each point represents triplicate values.
Effects of TRF,
-tocopherol and tocotrienols on the estrogen receptor.
Although MCF-7 cells are estrogen receptor-responsive, their growth is not estrogen dependent, and differences in plating density or growth medium can result in varied growth responses to estrogen (Soto and Sonnenschein 1985). To determine whether growth inhibition of MCF-7 cells by tocotrienols is acting through the estrogen receptor (ER) system, we treated the cells with the specified test compound at its IC50 concentration and examined the ability of estradiol to reverse this inhibition, using tamoxifen as a positive control. Table 3 shows that their inhibitory effect was not reversed by the addition of excess estradiol as occurs with tamoxifen. This indicates that their antiproliferative activity is probably not mediated through the ER.
|
Table 3. Inhibition of proliferation of MCF-7 cells in presence or absence of estrogen |
DISCUSSION
-tocopherol is ineffective. In the present study, we have shown that tocotrienols are effective inhibitors of both ER-negative and -positive cells and that combinations with tamoxifen produce a synergistic effect.
). However, most breast cancers are heterogeneous and consist of hormone-responsive as well as nonresponsive cells (Tiwari et al. 1991). A treatment regimen using a negative growth inhibitor coupled with an antihormonal drug would effectively target both types of tumor cells. Our results indicate that tocotrienols and tamoxifen have mutually potentiating effects on MDA-MB-435 cells and, in some cases, on MCF-7 cells, as measured by their effect on [3H] thymidine incorporation.
). Also, concerns have been raised that this drug may increase the incidence of new primary malignancies, such as endometrial, liver and colorectal cancers (Rutqvist et al. 1995). Because tocotrienols inhibit both ER+ and ER
cells, their potential for breast cancer treatment is of interest. The positive synergism observed with these compounds may allow the use of lower doses of tamoxifen, thus reducing the risk of side effects for patients.
). In smooth muscle cells, -tocopherol has been shown to inhibit protein kinase C (Chatelaine et al. 1993), which has been implicated in signal transduction and cellular proliferation (Nakamura and Nishizuka 1994). It is possible that tocotrienols might exert their antiproliferative properties by interfering with signal transduction events involving protein kinase C. The synergism between tocotrienols and tamoxifen indicates that they are acting by different mechanisms. Our data suggest that tocotrienols act via an ER-independent pathway. However, this does not rule out the involvement of the ER. Previous studies have shown that increased phosphorylation of the estrogen and progesterone receptors can alter their activity (Denner et al. 1990). Therefore, tocotrienols might exert their effect in part by interfering with the phosphorylation state.
- and -tocotrienols and tamoxifen were synergistic in MCF-7 cells.
FOOTNOTES
ACKNOWLEDGMENTS
This work was generously supported by the Palm Oil Research and Development Board of Malaysia. We thank Josephine Ho for excellent technical assistance and Charlotte Harman for assistance in typing the manuscript.
LITERATURE CITED
-tocopherol and -tocotrienol.
Rad. Biol. Med.
1991;
10:263-275, and tamoxifen in MCF-7 cells.
Cancer Lett.
1991;
61:45-52 [Medline]
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