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Effect of retinoic acid and palm oil carotenoids on oestrone sulphatase and oestradiol-17β hydroxysteroid dehydrogenase activities in MCF-7 and MDA-MB-231 breast cancer cell lines

2000; Wiley; Volume: 88; Issue: 1 Linguagem: Inglês

10.1002/1097-0215(20001001)88

1097-0215

Jin-Huat Ng, Kalanithi Nesaretnam, Karin Reimann, Leslie C. Lai,

Estrogen and related hormone effects

International Journal of CancerVolume 88, Issue 1 p. 135-138 Epidemiology and Cancer PreventionFree Access Effect of retinoic acid and palm oil carotenoids on oestrone sulphatase and oestradiol-17β hydroxysteroid dehydrogenase activities in MCF-7 and MDA-MB-231 breast cancer cell lines Jin-Huat Ng, Jin-Huat Ng Faculty of Medicine and Health Sciences, Universiti Putra Malaysia, Selangor, MalaysiaSearch for more papers by this authorKalanithi Nesaretnam, Kalanithi Nesaretnam Chemical and Technology Division, Palm Oil Research Institute of Malaysia (PORIM), Persiaran Institusi, Bandar Baru Bangi, Selangor, MalaysiaSearch for more papers by this authorKarin Reimann, Karin Reimann Faculty of Medicine and Health Sciences, Universiti Putra Malaysia, Selangor, MalaysiaSearch for more papers by this authorLeslie C. Lai, Corresponding Author Leslie C. Lai leslie@medic.upm.edu.my Faculty of Medicine and Health Sciences, Universiti Putra Malaysia, Selangor, MalaysiaFaculty of Medicine and Health Sciences, Universiti Putra Malaysia, 43400 UPM Serdang, Selangor, Malaysia. Fax: 603-89450130Search for more papers by this author Jin-Huat Ng, Jin-Huat Ng Faculty of Medicine and Health Sciences, Universiti Putra Malaysia, Selangor, MalaysiaSearch for more papers by this authorKalanithi Nesaretnam, Kalanithi Nesaretnam Chemical and Technology Division, Palm Oil Research Institute of Malaysia (PORIM), Persiaran Institusi, Bandar Baru Bangi, Selangor, MalaysiaSearch for more papers by this authorKarin Reimann, Karin Reimann Faculty of Medicine and Health Sciences, Universiti Putra Malaysia, Selangor, MalaysiaSearch for more papers by this authorLeslie C. Lai, Corresponding Author Leslie C. Lai leslie@medic.upm.edu.my Faculty of Medicine and Health Sciences, Universiti Putra Malaysia, Selangor, MalaysiaFaculty of Medicine and Health Sciences, Universiti Putra Malaysia, 43400 UPM Serdang, Selangor, Malaysia. Fax: 603-89450130Search for more papers by this author First published: 23 August 2000 https://doi.org/10.1002/1097-0215(20001001)88:1 3.0.CO;2-SCitations: 9AboutSectionsPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onFacebookTwitterLinked InRedditWechat Abstract Oestrogen is important in the development of breast cancer. Oestrogen receptor positive breast cancers are associated with a better prognosis than oestrogen-receptor negative breast cancers since they are more responsive to hormonal treatment. Oestrone sulphate acts as a huge reservoir for oestrogens in the breast. It is converted to the potent oestrogen, oestradiol (E2) by the enzymes oestrone sulphatase and oestradiol-17β hydroxysteroid dehydrogenase (E2DH). Retinoic acid and carotenoids have been shown to have chemopreventive activity against some cancers. The aim of our study was to determine and compare the effects of retinoic acid and palm oil carotenoids on growth of and oestrone sulphatase and E2DH activities in the oestrogen receptor positive, MCF-7 and oestrogen receptor negative, MDA-MB-231 breast cancer cell lines. Retinoic acid and carotenoids inhibited MCF-7 cell growth but had no effect on MDA-MB-231 cell growth. Both retinoic acid and carotenoids stimulated oestrone sulphatase activity in the MCF-7 cell line. E1 to E2 conversion was inhibited by 10−7 M carotenoids but was stimulated at 10−6 M in the MCF-7 cell line. Retinoic acid had no effect on E1 to E2 conversion at 10−7 M but stimulated E1 to E2 conversion at 10−6 M. Retinoic acid and carotenoids had no effect on E2 to E1 conversion in the MCF-7 cell line. Retinoic acid stimulated E1 to E2 conversion in the MDA-MB-231 cell line but had no effect on oestrone sulphatase activity or E2 to E1 conversion in this cell line. Both oestrone sulphatase and E2DH activity were not affected by carotenoids in the MDA-MB-231 cell line. In conclusion, retinoic acid and carotenoids may prevent the development of hormone-dependent breast cancers since they inhibit the growth of the MCF-7 cell line. Int. J. Cancer 88:135–138, 2000. © 2000 Wiley-Liss, Inc. Breast cancer is one of the leading causes of fatalities among women. Oestradiol (E2) is the most potent oestrogen and supports the growth of breast tumours (Duncan and Reed, 1995). Most breast cancers, about 95%, are initially oestrogen receptor positive but a substantial amount subsequently become oestrogen receptor negative and independent of oestrogen for growth (Henderson et al., 1988). The oestrogen receptor positive cancers have a better prognosis as they respond to hormonal treatment. Oestrone sulphate is significantly concentrated in breast cancer tissue compared with plasma levels (Pasqualini et al., 1997). Oestrone sulphate (E1S) forms a huge reservoir in breast tissues for the production of E2. The enzyme oestrone sulphatase converts E1S to active oestrone (E1). Oestradiol-17β hydroxysteroid dehydrogenase (E2DH) then converts E1 to the potent oestrogen, E2. E2DH catalyses both the reductive (E1 → E2) and oxidative (E2 → E1) reactions. The reductive activity of E2DH predominates in hormone dependent breast tumours (Poutanen et al., 1995). This gives rise to high concentrations of oestradiol in breast cancer tissues. Retinoids (e.g., retinoic acid) have been shown to affect breast cancer cell proliferation and differentiation (Zhu et al., 1997). Retinoids act through retinoic acid receptors (Pasquali et al., 1997). Although retinoids inhibit breast cancer growth, they increase the expression of E2DH Type 1 (Piao et. al.,1997). This leads to an increase in oestrogen production. Carotenoids consist of a large group of substances with various characteristics and biological functions, which are responsible for red-yellow colouration of fruits and vegetables (Olson, 1989). Carotenoids have pro-vitamin A and anti-oxidant property. They have been shown to possess chemopreventive activity against cancer cells (Murakoshi et. al., 1992). Palm oil carotenoids are destroyed during the refining process but a new refining technique has been developed to retain carotenoids in red palm oil (Choo et al., 1997). Palm oil carotenoids consist mainly of α and β-carotene: 41.9% and 51.7%, respectively (Choo et al., 1997; Cottrell, 1991). The effects of carotenoids on oestrone sulphatase and E2DH activities in breast cancer cell lines have not been studied previously. The aim of our study was to determine and compare the effects of both retinoic acid and palm oil carotenoids on cell growth and oestrone sulphatase and E2DH activities in the oestrogen receptor positive, MCF-7 and oestrogen receptor negative, MDA-MB-231, human breast cancer cell lines. MATERIAL AND METHODS [6,7 3H(N)]-E1S, ammonium salt (specific activity 53 Ci/mmol), [6,7 3H(N)]-E1 (40 Ci/mmol), [6,7 3H(N)]-E2 (48 Ci/mmol), 4-14C-E1 (57 mCi/mmol), 4-14C-E2 (53 mCi/mmol) were purchased from New England Nuclear Corp. (Boston, MA). The radioisotopes were purified before use. Caromin (palm oil carotenoid concentrate) was obtained from Carotech Sdn. Bhd. (Malaysia). Dulbecco's modified Eagle's medium (DMEM), RPMI 1640 and foetal bovine serum (FBS) were purchased from GibcoBRL Life Technologies, Inc. (Grand Island, NY). Unlabelled steroids E1 and E2 and all-trans retinoic acid were purchased from Sigma Chemical Co. (St. Louis, MO). Thin layer chromatography (TLC) sheets (Silica Gel 60 F254) were purchased from Merck (Darmstadt, Germany). Coulter Counter Z1, Zap-o-globin II and Isoton III used for cell counting were purchased from Coulter Corp. (Miami, FL). The liquid scintillation counter (Model 1217 RACKBETA LSC) was purchased from LKB (Wallag, Finland). Cell culture Both the MCF-7 and MDA-MB-231 cell lines were cultured in DMEM in 75 cm2 T-flask (Costar, Cambridge, MA). The MCF-7 cell line was supplemented with 5% (v/v) FBS, 2 mM L-glutamine and E2 at 10−8 M. The MDA-MB-231 cell line was supplemented only with 10% (v/v) FBS. All cell cultures were incubated at 37°C and 5% CO2. Prior to the experiments, DMEM was removed and replaced by RPMI 1640 containing 5% dextran/charcoal stripped FBS and 2 mM L-glutamine. The cells were grown in RPMI 1640 with stripped FBS and 2 mM L-glutamine for 2 to 3 days before the experiments. Treatment with retinoic acid and carotenoids For the experiments, cells were plated at a density of roughly 50,000 cells per well in a 12 well plate (Costar, Cambridge, MA) in 1 mL of RPMI 1640 stripped medium and incubated for 2–3 days at 37°C in 5% CO2 until half confluency. At the end of the incubation, the medium was discarded and retinoic acid (10−7 and 10−6 M) or carotenoids (10−7 and 10−6 M) were added. RPMI 1640 medium with stripped FBS was added to the control wells. The cells were grown for 3 days at 37°C and 5% CO2. All treatments and controls were done in triplicate. Oestrone sulphatase and E2DH assay Oestrone sulphatase and E2DH assays were carried out according to protocols previously described by Ee et al. (1999), Purohit et. al. (1992) and Adams et. al. (1988) with minor modifications. We found that cell numbers between 200,000 and 1,200,000 for the MCF-7 cell line and between 200,000 and 800,000 for the MDA-MB-231 cell line had a strong linear relationship with enzyme activity. These cell numbers were used for the experiments. At the end of the incubation, the medium was removed and the respective substrate added. For the oestrone sulphatase assay, 200,000 dpm 3H-E1S (1.7 pmol) was added while for the E2DH assay 200,000 dpm 3H-E1 (2.2 pmol; reductive assay) or 200,000 dpm 3H-E2 (1.9 pmol; oxidative assay) was added. The cells were incubated with the substrate for 4 hr. At the end of the incubation, the product formed was extracted using toluene (oestrone sulphatase assay) or diethyl ether (E2DH assay) and counted in a liquid scintillation counter. For the E2DH assay, the product was separated using TLC before the counting was done. Before the extraction, 50 μL of 14C-E1 (5,000 dpm) was added to correct for recovery in the oestrone sulphatase and oxidative E2DH assay. 14C-E2 (5,000 dpm) was used instead in the reductive E2DH assay. Results were expressed as fmol product/4 hr/106 cells. To determine the cell numbers, the cell monolayers were washed and incubated with Zap-o-globin before counting on a Coulter Counter. Statistical analysis Statistical analysis using unpaired Student's t-test was performed to determine differences between the cells treated with retinoic acid or carotenoids and the control cells. Results were considered to be statistically significant when p < 0.05. RESULTS Retinoic acid and palm oil carotenoids significantly inhibited MCF-7 cell growth at a concentration of 10−6 M (Fig. 1). The growth of MDA-MB-231 cells was not affected by retinoic acid or palm oil carotenoids over the range of concentrations tested. Oestrone sulphatase activity was significantly increased in the MCF-7 cell line by both retinoic acid and palm oil carotenoids at a concentration of 10−6 M (Fig. 2). In contrast, retinoic acid and carotenoids had no significant effect on oestrone sulphatase activity in the MDA-MB-231 cell line (Fig. 2). At 10−7 M carotenoids, the reductive E2DH activity (E1 to E2) was significantly inhibited in the MCF-7 cell line (Fig. 3). At a higher concentration, 10−6 M carotenoids, the reductive E2DH activity was significantly stimulated. On the other hand, 10−7 M retinoic acid did not have any effect on E1 to E2 conversion but significantly stimulated E1 to E2 conversion in the MCF-7 cell line at 10−6 M. Retinoic acid and carotenoids had no effect on oxidative E2DH activity (E2 to E1) in the MCF-7 cell line. In the MDA-MB-231 cell line, carotenoids did not have any significant effect on both oxidative and reductive E2DH activities but retinoic acid stimulated reductive E2DH activity at a concentration of 10−6 M (Fig. 4). All experiments were repeated twice and similar results were obtained. Figure 1Open in figure viewerPowerPoint Effect of a) retinoic acid and b) palm oil carotenoids on cell growth of MCF-7 and MDA-MB-231 cells. Cell counts are expressed as means of triplicate determinations + 1 standard deviation. ** p < 0.01, *** p < 0.001 vs. control. Figure 2Open in figure viewerPowerPoint Effect of a) retinoic acid and b) palm oil carotenoids on oestrone sulphatase activity in MCF-7 and MDA-MB-231 cell line. Oestrone sulphatase activities are expressed as means of triplicate determinations + 1 standard deviation. ** p < 0.01, *** p < 0.001 vs. control. Figure 3Open in figure viewerPowerPoint Effect of a) retinoic acid and b) palm oil carotenoids on reductive (E1 to E2) and oxidative (E2 to E1) E2DH activity in the MCF-7 cell line. Results are expressed as means of triplicate determinations + 1 standard deviation. * p < 0.05, ** p < 0.01 vs. control. Figure 4Open in figure viewerPowerPoint Effect of palm oil carotenoids on reductive (E1 to E2) and oxidative (E2 to E1) E2DH activity in the MDA-MB-231 cell line. Results are expressed as means of triplicate determinations + 1 standard deviation. * p < 0.05 vs. control. DISCUSSION The mean serum all-trans retinoic acid level has been shown to be 5.6 nmol/L and that of 13-cis retinoic acid level to be 5.8 nmol/L (Yeum et al., 1998). The concentrations used in the present study for retinoic acid are therefore supraphysiological, since physiological concentrations had no effect on cell growth. Plasma carotenoid concentrations have been found to be between 10−7 and 10−6 M (Ito et al., 1997) and is dependent on the dietary intake of carotenoids (Carughi et al., 1994). The carotenoid concentrations used in the present study are physiological. In the present study, both retinoic acid and carotenoids had anti-proliferative effects on the oestrogen dependent cell line, MCF-7, at 10−6 M. Retinoic acid and carotenoids had no effect on cell growth in the oestrogen independent cell line, MDA-MB-231. These findings concur with the results of previous studies looking at the effects of vitamin A on breast cancer cells (Zhu et al., 1997) and pro-vitamin A on liver (Narisawa et al., 1996) and lung (Kim et al., 1997) cancer cells. Previous studies have shown retinoic acid, a derivative of vitamin A, to be a strong inhibitor of breast cancer growth (Fontana, 1987; van der Burg et al., 1993). Carotenoids which are pro-vitamin A, for example α and β-carotene, can be converted into vitamin A to cause growth inhibition in oestrogen dependent MCF-7 cells. The importance of E1S in breast tumour progression is due to its conversion to E1 by the enzyme oestrone sulphatase (Santner et al., 1993). Retinoic acid and carotenoids stimulated oestrone sulphatase activity in the oestrogen dependent MCF-7 cell line. The stimulation of oestrone sulphatase activity results in the production of more E1, which can then be converted to the potent oestrogen E2. This would increase the risk of breast cancer. Oestrone sulphatase activity in the MDA-MB-231 cell line was not affected by the varying concentrations of retinoic acid or carotenoids tested. It is interesting to note that carotenoids at 10−7 M inhibited reductive E2DH activity, where this effect was not observed with the same concentration of retinoic acid. The mechanism of inhibition may be through an anti-oxidation effect of carotenoids on co-factors, which are necessary for E2DH activity. The inhibition of E2DH activity in the MCF-7 cell line was, however, not accompanied by growth inhibition. It has been shown that addition of oxidised co-factor NAD+ increased the activity of E2DH and reduced availability of this co-factor decreased E2DH activity in the oestrogen dependent MCF-7 (Duncan and Reed, 1995) and T-47D (Reed et al.,1994) breast cancer cell lines. Reed et al. (1994) noted that the availability of the co-factors was not found to be limiting but governed the direction (reductive or oxidative) in which E2DH acts. At a higher carotenoid concentration of 10−6 M, the reductive E2DH activity in the MCF-7 cell line was stimulated. Retinoic acid at this concentration also stimulated reductive E2DH activity. The stimulation of reductive E2DH activity was accompanied by growth inhibition. Both retinoic acid and carotenoids had no effect on oxidative E2DH activity in the MCF-7 cell line. Stimulation of reductive E2DH activity while having no effect on oxidative E2DH activity would increase the amount of the potent oestrogen, E2, which would normally increase the proliferation of the MCF-7 cell line. Since growth inhibition was observed it can be surmised that the effect of carotenoids on growth of the MCF-7 cell line is via a different pathway from that of E2-stimulated MCF-7 cell growth. In the MDA-MB-231 cell line, retinoic acid stimulated reductive but not oxidative E2DH activity, while both reductive and oxidative E2DH activities were not affected by carotenoids. Since oestrogens do not have an effect on cell growth of the MDA-MB-231 cell line the ability of retinoic acid to increase E1 to E2 conversion in this cell line is of no biological significance. 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