- Email: [email protected]
Effect of vitamin E on tamoxifen-treated breast cancer cells
Effect of vitamin E on tamoxifen-treated breast cancer cells Elizabeth A. Peralta, MD, Melita L. Viegas, MD, Somaja Louis, MS, Deborah L. Engle, MS, and Gary L. Dunnington, MD, Springfield, Ill
Background. Induction of apoptosis by tamoxifen has been postulated to involve oxidative stress. Tamoxifen (TAM) may act on estrogen receptors (ER) located in the plasma membrane. Our hypothesis that supplemental antioxidant vitamin E (␣-tocopherol) acts at the plasma membrane to alter the effectiveness of tamoxifen was tested in ER-positive breast cancer cell lines, MCF-7 and T47D. Methods. Cells were treated in vitro with 20-M TAM alone and in combination with 10-M ␣-tocopherol (AT). Estrogen growth signals were quantified by immunohistochemical staining for the mitogen-activated protein kinase p-ERK. Rapid changes in intracellular calcium were detected in TAM-treated MCF-7 and T-47D cells by fluorescence microscopy of cells loaded with the calciumsensitive dye Fluo 4AM. Apoptosis was assayed by flow cytometry. Results. Proliferating cells in normal medium exhibited strong p-ERK staining. Addition of TAM abolished p-ERK staining and caused cell rounding and death. The addition of AT led to the restoration of cell proliferation and p-ERK expression even in the presence of high-dose TAM. Intracellular calcium rapidly increased in MCF-7 and T47D cells upon exposure to TAM, followed by an increase in caspase activation and eventual apoptosis. The increase in intracellular calcium was abolished by the addition of 10M AT to TAM, and pan-caspase staining decreased at 5 hours from 72% to 41%. Conclusions. These studies suggest that supplemental vitamin E decreases the inhibitory effect of TAM on the proliferation of ER⫹ breast cancer cells and eliminates the rapid rise in intracellular calcium that leads to apoptosis stimulated by TAM. The use of vitamin E acetate supplements may be inadvisable for women taking tamoxifen. (Surgery 2006;140:607-15.) From the Department of Surgery, Southern Illinois University School of Medicine, Springfield, Ill
A milestone in the adjuvant treatment of breast cancer was the demonstration of disease-free and overall survival benefit conferred by the selective estrogen receptor modulator, tamoxifen.1-3 Tamoxifen remains a commonly prescribed drug for treatment and prevention of hormone-responsive breast cancer. The mechanism of action of tamoxifen is through binding with the estrogen receptor-␣ (ER) and inhibiting the transcriptional activation of estrogen response elements in the Presented at the 63rd Annual Meeting of the Central Surgical Association, Louisville, Kentucky, March 9-11, 2006. Supported by the Illinois Department of Public Health Penny Severns Breast and Cervical Cancer Research Fund and the Breast Center at SIU Breast Cancer Research Fund. Accepted for publication July 10, 2006. Reprint requests: Elizabeth A. Peralta, MD, Southern Illinois University School of Medicine, P.O. Box 19638, Springfield, IL 62794-9638. E-mail: [email protected]. 0039-6060/$ - see front matter © 2006 Mosby, Inc. All rights reserved. doi:10.1016/j.surg.2006.07.007
genome.4 There has been increasing evidence for a role of estrogen receptors located in the plasma membrane in the signaling activity of tyrosine kinase receptors, such as the epidermal growth factor receptors EGFR, HER-1, and Her-2 neu.5 In breast cancers, an inverse correlation has been noted between the level of expression of ER and HER-2 neu and other EGFRs. Increased EGFR expression also has been implicated in the development of tamoxifen resistance.6,7 Similarly for tamoxifen, a variety of nongenomic pathways may contribute to the antitumor effect, possibly mediated by signaling events occurring at the cell surface. These signals include modulation of protein kinase C, calmodulin, TGF-, ceramide, activated JNK, p38, and mitochondrial toxicity with collapse of the transmembrane potential and caspase activation.8 The in vitro induction of apoptosis by tamoxifen has been postulated to involve oxidative stress.9 Given the possible importance of oxidative stress in the antitumor effect of tamoxifen, antioxidant vitamin supplementation may be counterproducSURGERY 607
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tive. 〈lpha-tocopherol acetate, the popular form of vitamin E supplement taken by many healthy adults and cancer patients, also is considered to be a harmless remedy for hot flashes. A prospective, blinded, placebo-controlled trial with crossover compared the benefit of 800 IU/day vitamin E versus placebo in women with vasomotor symptoms.10 The study involving 105 patients showed that vitamin E was associated with a minimal decrease in hot flashes (1 less hot flash per day than was seen with placebo) (P ⱕ .05). Megadose vitamin E differs from a placebo in that it influences the biochemical milieu in which other cancer therapies are acting. It has its most important function as a free-radical scavenger in the lipid compartment of cells and serum, where increased serum vitamin E levels correspond to decreased lipid peroxidation. It is a potent inhibitor of protein kinase C and 5-lipoxygenase.11,12 In vitro, vitamin E also inhibits smooth muscle cell proliferation, platelet aggregation, and the oxygen burst in neutrophils.13,14 The hypothesis that supplemental vitamin E (␣-tocopherol [AT]) alters the effectiveness of tamoxifen was tested using in vitro models of human breast carcinoma. One approach to demonstrating a nongenomic mechanism for tamoxifen effect is to assess the activation of plasma membrane–associated signaling. Intracellular calcium concentration [Ca 2⫹]i is a highly versatile second messenger in the cell involved in motility, growth, differentiation, and apoptosis.15 The steep concentration gradient between extracellular calcium and [Ca 2⫹]i allows small changes to trigger Ca2⫹-dependent events. Both estrogen and tamoxifen have been observed causing rapid rises in [Ca 2⫹]i in a variety of cultured cells including vascular endothelial cells, chicken granulosa cells, and breast cancer cells.16-18 In the present study, we measured rapid increases in [Ca 2⫹]i in ER⫹ cancer cell lines upon exposure to tamoxifen, and we related the timing and culture conditions to the onset of apoptosis. Phosphorylation of the mitogen-activated protein (MAP) kinases ERK-1 and ERK-2 has been shown to be stimulated by estradiol in the ER⫹ breast cancer cell line MCF-7. This p-ERK expression is associated with increased proliferation and is blocked by the ER antagonist ICI 182,780.19 In MCF-7, p-ERK expression is reduced by tamoxifen.20 Paired specimens of ER⫹ breast cancers before and after progression during tamoxifen treatment showed increased expression of activated p38 and p-ERK.21 Therefore, we have used p-ERK expression combined with cell proliferation as an indication of tamoxifen resistance.
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MATERIALS AND METHODS Cell lines. Human breast cancer cell lines MCF-7, T47D, and MDA MB 231 were obtained from the American Type Culture Collection (ATCC, Manassas, Va). The prostate cancer cell line LNCap was a kind gift from Dr Louis Premkumar. Cells were screened negative for mycoplasma and maintained in Dulbecco modified Eagle medium (DMEM) supplemented with 10% fetal bovine serum (FBS), 200 mM glutamine and penicillin. In vitro proliferation assays. MCF-7 and T47D cells were plated at 5000 cells/well in a 96-well plate and incubated overnight at 37°C and 5% CO2 in normal medium before challenge with ␣-tocopherol (AT) (10-100 M) alone and in combination with 10 M tamoxifen (TAM) in MCF-7 and T47D cells for 24 hours. MTS substrate (Cell Titre96 Aqueous Non-radioactive Cell Proliferation Assay, Promega Corp, Madison, Wis) was then added. Plates were incubated for 3 hours and spectrometry was performed to read absorbance at 490 nm. MCF-7 and T47D assays were run in triplicate and repeated in 3 separate experiments. Immunohistochemistry. T47D cells and MCF-7 cells were grown on sterile chambered slides with different doses of tamoxifen (10, 20, and 40M) and in combination with tamoxifen (40 M) and different doses of AT (10, 50, and 100M) for 24 hours. The slides were fixed in 4% methanol-free formaldehyde for 25 minutes at 4°C and then processed for immunohistochemistry using R.T.U Vectastain Universal ABC kit (Vector Laboratories, Burlingame, Calif). A mouse monoclonal pERK antibody (Santa Cruz Biotechnology, Inc., Santa Cruz, Calif) was used at 1:100 dilution. The cells were incubated overnight at 4°C. Negative controls were incubated with phosphate-buffered saline (PBS) for the same period. After washing, the slides were incubated for 30 minutes in biotinylated universal secondary antibody and stained according to the manufacturer’s protocol. For each slide, p-ERK staining was scored as 0 if no cells exhibited deep brown cytoplasmic staining, 1⫹ if 10% of cells were stained, and 2⫹ if more than 10% of cells were stained. Intracellular calcium imaging. The estrogen receptor-positive (ER⫹) breast cancer cell lines MCF-7 and T47D, and the estrogen receptor ␣-negative cell lines MDA-MB-231(breast carcinoma) and LNCap (an estrogen receptor -positive prostate carcinoma) were tested in normal culture medium, steroid-free medium, and medium containing 10 M AT. Cells were loaded with the
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Fig 1. Proliferation of breast cancer cells treated with tamoxifen and vitamin E. a, Effect of different doses of ␣-tocopherol (AT) and tamoxifen (TAM) at 24 hours incubation on proliferation of T47.D breast tumor cells; AT 10 M or 50 M alone had no effect. Tamoxifen-treated cells displayed a dose-dependent decrease in proliferation rates when compared to cells treated with media and vehicle. Combination treatment of MCF-7 (b) and T47D ER⫹ (c) breast cancer cell lines with 40uM TAM and various concentrations of AT displayed significantly higher proliferation rates compared to cells treated with TAM alone. *P ⬍ .005.
calcium ion (Ca2⫹)-sensitive dye Fluo 4AM (Molecular Probes, Eugene, Ore) and then transferred to a reading chamber with continuous flow of test medium. Fluo 4AM was excited at 488 nm, and the emitted fluorescence of single cells was filtered with a 535 ⫾ 25 nm bandpass filter and read into a computer using Scanalytics software (Scanalytics, Fairfax, Va). Changes in the intensity of fluorescence (F/Fo) were determined using fluorescence microscopy (DMIRE2; Leica, Plymouth, Minn) and plotted against time to evaluate changes in intracellular calcium levels [Ca 2⫹]i in the presence of TAM and TAM plus AT. Thapsigargin is an inhibitor of the Ca2⫹ ATPase that stores Ca2⫹ in the smooth endoplasmic reticulum. Thapsigargin treatment was used to test for an influx of extracellular Ca2⫹ through channels distinct from voltage-gated Ca2⫹ channels, a process termed capacitative Ca2⫹ entry.22 Apoptosis assays. MCF-7 cells were pretreated for 1 hour with 0 or 10M AT in normal medium before the addition of 20 M TAM. Apoptosis was quantified at 0, 2, and 5 hours using fluorescence-activated cell sorting and staining with FITC-VD-FMK (ApoStat; R&D Systems, Minneapolis, Minn), an indicator of pan-caspase activity. RESULTS Alpha tocopherol increases the proliferation rate of TAM-treated cells. TAM-treated T47D cells displayed a dose-dependent decrease in proliferation rate compared with cells treated with media or vehicle, with a 95% inhibition at 40 M TAM (Fig 1, a). Addition of AT to media containing 40 M TAM restored the proliferation rate to 60% of media control (Fig 1, b). In MCF-7 cells, 40 M TAM
Fig 2. Effect of AT on cell number and morphology in TAM-treated cells. a, T47D cells cultured on chambered slides in 40 M TAM alone and with 50 M AT. b, MCF7 cells cultured on chambered slides in 40 M TAM alone and with 50 M AT (Hematoxylin/eosin stain; magnification: X200).
reduced proliferation by 68%. Addition of AT to 40 M TAM restored the proliferation rate from 32% to 100% of media control (Fig 1, c). The effect was significant at all concentrations of AT, but was maximal in the range of 10 to 100 M. Culturing cells under these media conditions on chambered slides and staining with hematoxylin/eosin shows scarce, pyknotic cells with TAM treatment compared with confluent, viable cells exposed to TAM⫹AT (Fig 2, a and b). These findings demon-
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Fig 3. Effect of AT on p-ERK expression in TAM-treated cells. a, MCF-7 cells negative control; b, MCF-7 cells in normal medium (positive control); c, MCF-7 cells treated for 24 hours with Tam 40 M; d, MCF-7 cells treated with combination of Tam 40 M and AT 50 M. e, T47D cells negative control; f, T47D cells in normal medium (positive control); g, T47D cells treated for 24 hours with Tam 40 M; h, T47D cells treated with combination of Tam 40 M and AT 50 M. (p-ERK immunostain; magnification: X200).
strate that AT can cause tamoxifen resistance in hormone-sensitive cells. P-ERK expression is restored in TAM-treated cells exposed to alpha-tocopherol. MCF-7 and T47D both express p-ERK when growing in normal medium that contains estrogen from fetal calf serum (Fig 3, b and f). P-ERK expression was decreased to 0-1⫹ in TAM-treated cells (Fig 3, c and g). Addition of AT to TAM restored expression of p-ERK to 2⫹ and increased the number and viability of both MCF7 and T47D cells (Fig 3, d and h). From these experiments, it is postulated that p-ERK expression is associated with TAM-resistance in hormone-sensitive breast cancer cells. Alpha tocopherol prevents the increase of intracellular calcium, a signal preceding apoptosis of ERⴙ tumor cells. Exposure to TAM (5 to 20M) stimulated a rapid (onset at 4 minutes) and significant increase in [Ca 2⫹]i in T47D and MCF-7 cells (Fig 4, a and b). The signal was self-limiting, reproducible, and was stronger with greater TAM dose (Fig 4, c). A striking finding was that the increase in [Ca 2⫹]i was abolished by flowing medium containing AT over the cells for 15 minutes between TAM exposures and by preincubation with AT for 24 hours prior to TAM treatment (Fig 4, d and e). Removing Ca2⫹ from the bathing medium abolished the rise in [Ca 2⫹]i. With Ca2⫹ removed from the extra cellular medium, intracellular Ca2⫹ stores are not released by TAM, as demonstrated by the transient rise in [Ca 2⫹]i when thapsigargin is added after TAM (Fig 4, f). These experiments indicate that the increase in concentration of intracellular calcium in MCF-7 and T47D cells is not by release of Ca2⫹
from endoplasmic reticulum stores and requires Ca2⫹entry from the extracellular medium. ER expression was necessary for TAM to elicit the [Ca 2⫹]i signal. TAM did not stimulate an increase in [Ca 2⫹]i in the estrogen receptor ␣-negative cell lines MDA-MB-231, LNCap, or MCF-7 cells that had been incubated in steroid-free medium to reduce ER expression (Fig 4, g-i). These results support a link between the absence of TAM-stimulated intracellular calcium flux and tamoxifen-resistance. Alpha-tocopherol decreases tamoxifen-induced apoptosis in vitro in ERⴙ breast cancer cell lines. MCF-7 cells are defective for caspase-3, a rapid effector caspase, but MCF-7 cells can undergo activation of other caspases and morphologic changes of apoptosis with TAM treatment with an onset of 90 minutes.23 We used a pan-caspase fluorescent substrate to quantify apoptosis by fluorescenceactivated cell sorting. The population of apoptotic MCF-7 cells at 5 hours was 72% in cells treated with 20 M TAM alone (Fig 5, a) compared with 41% in cells treated with TAM plus 10 M AT (Fig 5, b). DISCUSSION Cancer patients frequently report use of vitamin E supplements during and after therapy. A casecontrol study in which interviews with Wisconsin women aged 18 to 69 years who were recent breast cancer survivors were compared with interviews of randomly selected controls found that cancer survivors were 5.3 times more likely than controls to experience menopausal symptoms and 7.4 times more likely to use alternative remedies such as soy, vitamin E, or herbs.24 Because the mechanism of
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Fig 4. Effect of tamoxifen on intracellular calcium (Cai). a, T47.D Breast cancer cells; b, MCF-7 breast cancer cells. c, Effect of different doses of tamoxifen 5-M and 20-M doses on T4.7 cells. Bar indicates the time of Tam addition. Individual cell tracings recorded simultaneously are shown. d, The increase in Cai after exposure to tamoxifen 20 uM was abolished by 10 M AT in T47.D cells by flowing ␣-tocopherol acetate between Tamoxifen exposures. e, In MCF-7 cells, preincubation with AT for 24 hours abolished the rise in Cai when Tam is added. f, Effect of removing extracellular Ca2⫹ on intracellular calcium stores (a) 1 uM Thapsigargin and 20 um Tamoxifen were added as indicated. After releasing smooth endoplasmic reticulum calcium stores with thapsigargin, administration of Tamoxifen did not cause an increase in Cai. g, Effect of Tam dose on Cai in ER-negative MDA-MB-231 cells; h, same for ER ␣-negative, ER -positive LNCap cells (prostate); i, same for MCF-7 cells cultured in steroid-free medium to decrease ER expression. Tamoxifen displayed minimal or no effect in these cells.
action of certain therapies such as radiation and chemotherapy involve the generation of oxygen radicals, it is a matter of concern whether antioxidant supplements should be used. Although the magnitude of the in vitro effect of AT in this study was large, it may not necessarily
reflect the in vivo impact of dietary AT. We supplemented the cell culture medium with vitamin E to concentrations of 10 to 50 M. The normal human serum level of vitamin E ranges from 11.6 to 38 M and is dependent on serum lipid levels and activity of AT transfer protein in the liver. The recom-
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Fig 5. Vitamin E reduces tamoxifen-induced apoptosis in vitro. MCF-7 cells were pretreated for 1 hour with 0 (a) or 10 (b) M AT before the addition of 20 M TAM. Apoptosis is shown at 5 hours using flow cytometry and staining with FITC-VD-FMK (R&D Systems), an indicator of pan-caspase activity.
mended daily allowance of vitamin E is 15 mg/day compared to the average over-the-counter vitamin E capsule, which contains 400 mg. When supplements are taken, the serum level does not predictably rise. A study of volunteers given a dose of 75 mg natural AT resulted in a plasma concentration rise at 12 hours from 0.3 to 12.4 M.25 The Nurses Health Study, a cohort of 83,234 women enrolled in 1976, has been followed by Zhang et al for multivariate analyses of breast cancer risk and lifestyle factors including dietary intakes of carotenoids and vitamins A, C, and E.26 Whereas consumption of fruits and vegetables high in specific carotenoids was associated with a mild reduction in breast cancer risk in premenopausal women, total vitamin E from food and supplements was related to a weak increased risk of premenopausal breast cancer. Zhang and colleagues speculated that perhaps women with worse fibrocystic breast disease might have been more likely to take vitamin E supplements. Given that vitamin E is not contributory to breast cancer prevention and may antagonize the effect of tamoxifen, it seems inadvisable for women who take tamoxifen for chemprevention of breast cancer also take vitamin E supplements. An important clinical use of tamoxifen is in the adjuvant setting, ie, in women with no evidence of disease. In such a setting, it is difficult to assess the effect of vitamin E on tamoxifen unless a difference in relapse rate can be observed. In a prospective study of 363 patients newly diagnosed with breast cancer, blood was collected prior to therapy to determine levels of vitamin E, plasma cholesterol, lipoperoxides, and glutathione oxidase. There was an inverse relationship between tumor size and plasma lipoperoxides, and higher lipoperoxide levels were associated with lower-stage (node-negative) disease. A total of 66% of the patients had ER⫹ tumors and 89% of these received adjuvant hormonal therapy. With a median follow-up of 8 years, they found an increased risk of
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recurrence ([RR] 1.7) and breast cancer death (RR 2.0) in patients with plasma-lipid adjusted vitamin E levels above 22 mol/L.27 Other studies have found that higher vitamin E intake correlated with increased treatment failure within 2 years in women with ER⫹ breast cancer.28 An interaction between tamoxifen and vitamin E in breast cancer cell lines has been previously observed. Tamoxifen caused growth inhibition via downregulation of protein kinase C in ER-negative MDA MB 231 cells.9 In that experiment, TAM was found to partition in the plasma membrane, and the growth inhibition was abolished by antioxidants such as vitamin E and superoxide dismutase. In other ER-negative cells, TAM induced apoptosis through JNK1 and caspase-3 activation. Apoptosis was blocked by vitamin E but not the water-soluble antioxidants N-acetyl L-cysteine and glutathione.29 In contrast, nonantioxidant vitamin E analogues such as the tocotrienols and ␣-tocopherol succinate (␣-TOS) have a proapoptotic effect on breast and other cancer cell lines.30-32 Multiple signaling pathways have been implicated including activation of the caspase 3 cascade, inhibition of PKC␣, and activation of JNK1.33 In animal models, ␣-TOS had to be administered by intraperitoneal injection; oral administration leads to hydrolysis of ␣-TOS to AT, which had no antitumor effect. These observations of nonantioxidant vitamin E analogues are not inconsistent with our hypothesis that the antioxidant effect of vitamin E in the plasma membrane may interfere with tamoxifen-induced apoptosis. The present findings suggest that the mechanism for vitamin E–induced tamoxifen resistance involves interruption of plasma membrane ER signaling via blocking a Ca2⫹ second messenger and increasing p-ERK activation. Recently, an estrogen receptor of a different class, a G protein-coupled receptor (GPR30), has been described.34 GPR30 may have a crucial role in the estrogenstimulated activation of p-ERK. It was observed that 17beta-estradiol activates ERK-1/-2 in MCF-7 cells, which express both estrogen receptor alpha (ER alpha) and ER beta, and in SKBR3 breast cancer cells, which fail to express either receptor. Immunoblot analysis using GPR30 peptide antibodies showed that this estrogen response was associated with the presence of GPR30 protein in these cells.35 GPR30 binds not only 17beta-estradiol but also 1tamoxifen and ICI 182,780 with an agonist signal mimicking estrogen.36 This G-protein activation results in the activation of cascades of signal transduction including mobilization of intracellular calcium, production of cAMP, stimulation of phosphlipase C, protein kinase C, and other proteins. It
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is unknown how vitamin E may affect GPR30 signaling. A hypothesis that fits our findings to date is that vitamin E may block the proapoptotic Ca2⫹ signal while TAM binding to GPR30 activates p-ERK, thus giving a net growth-stimulatory effect of TAM at the cell membrane and contributing to tamoxifen-resistance. The efficacy of tamoxifen as it is used in practice, ie, in combination with vitamin supplements, might be assessable with surrogate endpoint biomarkers. Candidate biomarkers are Ki67, ER, PR, and p-ERK in ductal epithelial cells from normal breast biopsies of patients in adjuvant therapy. We are currently conducting a pilot study of these biomarkers in women who are already taking vitamin E concurrent with tamoxifen and are willing to undergo random core breast biopsies 4 weeks before and after they discontinue vitamin E. Such a study may provide insight into the interaction of oxidative status and selective estrogen receptor modulation and identify useful biomarkers to assess other prevention drugs. In the meanwhile, it appears prudent to discourage vitamin E supplementation in women taking tamoxifen. The authors thank Dr. Sophia Ran for editorial assistance.
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REFERENCES 1. Fisher B, Redmond C, Legault-Poisson S, Dimitrov NV, Brown AM, Wickerham DL, et al. Postoperative chemotherapy and tamoxifen compared with tamoxifen alone in the treatment of positive-node breast cancer patients aged 50 years and older with tumors responsive to tamoxifen: results from the National Surgical Adjuvant Breast and Bowel Project B-16. J Clin Oncol 1990;8:1005-18. 2. Fisher B, Dignam J, Bryant J, DeCillis A, Wickerham DL, Wolmark N, et al. Five versus more than five years of tamoxifen therapy for breast cancer patients with negative lymph nodes and estrogen receptor-positive tumors. J Natl Cancer Inst 1996;88:1529-42. 3. Early Breast Cancer Trialists’ Collaborative Group. Effects of adjuvant tamoxifen and of cytotoxic therapy on mortality in early breast cancer. An overview of 61 randomized trials among 28,896 women. N Engl J Med 1988;319:1681-92. 4. MacGregor JI, Jordan VC. Basic guide to the mechanisms of antiestrogen action. Pharmacol Rev 1998;50:151-96. 5. Schiff R, Massarweh SA, Shou J, Bharwani L, Mohsin SK, Osborne CK. Cross-talk between estrogen receptor and growth factor pathways as a molecular target for overcoming endocrine resistance. Clin Cancer Res 2004;10:331S-6S. 6. Gee JM, Robertson JF, Ellis IO, Nicholson RI. Phosphorylation of ERK1/2 mitogen-activated protein kinase is associated with poor response to anti-hormonal therapy and decreased patient survival in clinical breast cancer. Int J Cancer 2001;95:247-54. 7. Shou J, Massarweh S, Osborne CK, Wakeling AE, Ali S, Weiss H, et al. Mechanisms of tamoxifen resistance: in-
17.
18.
19.
20.
21.
22.
23.
creased estrogen receptor-HER2/neu cross-talk in ER/HER2-positive breast cancer. J Natl Cancer Inst 2004;96:926-35. Mandlekar S, Kong AN. Mechanisms of tamoxifen-induced apoptosis. Apoptosis 2001;6:469-77. Gundimeda U, Chen ZH, Gopalakrishna R. Tamoxifen modulates protein kinase C via oxidative stress in estrogen receptor-negative breast cancer cells. J Biol Chem 1996;271: 13504-14. Barton DL, Loprinzi CL, Quella SK, Sloan JA, Veeder MH, Egner JR, et al. Prospective evaluation of vitamin E for hot flashes in breast cancer survivors. J Clin Oncol 1998;16:495-500. Ricciarelli R, Zingg JM, Azzi A. Vitamin E: protective role of a Janus molecule. Faseb J 2001;15:2314-25. Pratico D, Tangirala RK, Rader DJ, Rokach J, FitzGerald GA. Vitamin E suppresses isoprostane generation in vivo and reduces atherosclerosis in ApoE-deficient mice. Nat Med 1998;4:1189-92. Brigelius-Flohe R, Kelly FJ, Salonen JT, Neuzil J, Zingg JM, Azzi A. The European perspective on vitamin E: current knowledge and future research. Am J Clin Nutr 2002;76:703-16. Wesselborg S, Bauer MK, Vogt M, Schmitz ML, SchulzeOsthoff K, et al. Activation of transcription factor NFkappaB and p38 mitogen-activated protein kinase is mediated by distinct and separate stress effector pathways. J Biol Chem 1997;272:12422-9. Krebs J. The role of calcium in apoptosis. Biometals 1998; 11:375-82. Rubio-Gayosso I, Sierra-Ramirez A, Garcia-Vazquez A, Martinez-Martinez A, Munoz-Garcia O, Morato T, et al. 17Betaestradiol increases intracellular calcium concentration through a short-term and nongenomic mechanism in rat vascular endothelium in culture. J Cardiovasc Pharmacol 2000; 36:196-202. Morley P, Whitfield JF, Vanderhyden BC, Tsang BK, Schwartz JL. A new, nongenomic estrogen action: the rapid release of intracellular calcium. Endocrinology 1992;131: 1305-12. Chang HT, Huang JK, Wang JL, Cheng JS, Lee KC, Lo YK, et al. Tamoxifen-induced increases in cytoplasmic free Ca2⫹ levels in human breast cancer cells. Breast Cancer Res Treat 2002;71:125-31. Migliaccio A, Di Domenico M, Castoria G, de Falco A, Bontempo P, Nola E, et al. Tyrosine kinase/p21ras/MAPkinase pathway activation by estradiol-receptor complex in MCF-7 cells. Embo J 1996;15:1292-300. Yang D, Wang S, Ye Y, Cui Z. [Effect of mitogen-activated protein kinase signal transduction in non-estrogen antagonistic mechanism of tamoxifen]. Zhonghua Yi Xue Za Zhi 2002;82:1232-4. Gutierrez MC, Detre S, Johnston S, Mohsin SK, Shou J, Allred DC, et al. Molecular changes in tamoxifen-resistant breast cancer: relationship between estrogen receptor, HER-2, and p38 mitogen-activated protein kinase. J Clin Oncol 2005;23:2469-76. Hunton DL, Lucchesi PA, Pang Y, Cheng X, Dell’Italia LJ, Marchase RB. Capacitative calcium entry contributes to nuclear factor of activated T-cells nuclear translocation and hypertrophy in cardiomyocytes. J Biol Chem 2002;277: 14266-73. Rehm M, Dussmann H, Janicke RU, Tavare JM, Kogel D, Prehn JH. Single-cell fluorescence resonance energy transfer analysis demonstrates that caspase activation during ap-
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24.
25.
26.
27.
28.
29.
30.
31. 32.
33.
34.
35.
36.
optosis is a rapid process. Role of caspase-3. J Biol Chem 2002;277:24506-14. Harris PF, Remington PL, Trentham-Dietz A, Allen CI, Newcomb PA. Prevalence and treatment of menopausal symptoms among breast cancer survivors. J Pain Symptom Manage 2002;23:501-9. Roxborough HE, Burton GW, Kelly FJ. Inter- and intraindividual variation in plasma and red blood cell vitamin E after supplementation. Free Radic Res 2000;33:437-45. Zhang S, Hunter DJ, Forman MR, Rosner BA, Speizer FE, Colditz GA, et al. Dietary carotenoids and vitamins A, C, and E and risk of breast cancer. J Natl Cancer Inst 1999;91:547-56. Saintot M, Mathieu-Daude H, Astre C, Grenier J, SimonyLafontaine J, Gerber M. Oxidant-antioxidant status in relation to survival among breast cancer patients. Int J Cancer 2002;97:574-9. Holm LE, Nordevang E, Hjalmar ML, Lidbrink E, Callmer E, Nilsson B. Treatment failure and dietary habits in women with breast cancer. J Natl Cancer Inst 1993;85:32-6. Mandlekar S, Yu R, Tan TH, Kong AN. Activation of caspase-3 and c-Jun NH2-terminal kinase-1 signaling pathways in tamoxifen-induced apoptosis of human breast cancer cells. Cancer Res 2000;60:5995-6000. Neuzil J, Weber T, Schroder A, Lu M, Ostermann G, Gellert N, et al. Induction of cancer cell apoptosis by alpha-tocopheryl succinate: molecular pathways and structural requirements. Faseb J 2001;15:403-15. Malafa MP, Neitzel LT. Vitamin E succinate promotes breast cancer tumor dormancy. J Surg Res 2000;93:163-70. Guthrie N, Gapor A, Chambers AF, Carroll KK. Inhibition of proliferation of estrogen receptor-negative MDA-MB-435 and -positive MCF-7 human breast cancer cells by palm oil tocotrienols and tamoxifen, alone and in combination. J Nutr 1997;127:544S-8S. Yu W, Simmons-Menchaca M, You H, Brown P, Birrer MJ, Sanders BG, et al. RRR-alpha-tocopheryl succinate induction of prolonged activation of c-jun amino-terminal kinase and c-jun during induction of apoptosis in human MDA-MB-435 breast cancer cells. Mol Carcinog 1998;22:247-57. Carmeci C, Thompson DA, Ring HZ, Francke U, Weigel RJ. Identification of a gene (GPR30) with homology to the G-protein-coupled receptor superfamily associated with estrogen receptor expression in breast cancer. Genomics 1997;45:607-17. Filardo EJ, Quinn JA, Bland KI, Frackelton AR Jr. Estrogen-induced activation of Erk-1 and Erk-2 requires the G protein-coupled receptor homolog, GPR30, and occurs via trans-activation of the epidermal growth factor receptor through release of HB-EGF. Mol Endocrinol 2000;14: 1649-60. Thomas P, Pang Y, Filardo EJ, Dong J. Identity of an estrogen membrane receptor coupled to a G-protein in human breast cancer cells. Endocrinology 2005;146:624-32.
DISCUSSION Dr Aness Chagpar (Louisville, Ky): In this study, Dr Peralta and her colleagues from the Southern Illinois University School of Medicine investigate the effect of combining alpha-tocopherol acetate, a component of vitamin E, with tamoxifen on breast cancer cell line growth, apoptosis and intracellular signaling.
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In an era of growing interest in alternative and complementary therapies, their work is particularly timely. They demonstrated that the addition of alpha-tocopherol decreased the inhibitory effects of tamoxifen on the proliferation of estrogen receptor positive breast cancer cells, and, furthermore, that it eliminated the rapid rise in intracellular calcium leading to apoptosis. A number of investigators have found that alphatocopherol may blunt the inhibitory effect of tamoxifen. Tocotrienols, however, which are another component of vitamin E formulation, have not been shown to have the same effect in some studies. Did you look at tocotrienols and could you comment on what could account for the difference between the tocotrienols and alpha-tocopherol? What is the dose of vitamin E that we get with oral supplementation and how does that tie into your results? There is newer data suggesting that aromatase inhibitors, which act through a different mechanism through peripheral conversion, may be more efficacious than tamoxifen in postmenopausal women. Given these data, could you speculate on what effect you might expect with vitamin E derivatives and their effect on aromatase inhibitors? Dr Gerald M. Larson (Louisville, Ky): Could you tell us how large the subset of patients is that takes both vitamin E and tamoxifen? Dr Elizabeth A. Peralta: The subset of people we are interested in are women who are taking tamoxifen either for breast cancer adjuvant therapy or prevention. In my practice following up with patients, I would ask them what they are taking in addition to their prescription medications, and I found that vitamin E was rather common. What I have learned in looking at some of the other components of vitamin E is that the nonantioxidant forms of vitamin E such as the tocotrienols have an antitumor or proapoptotic effect on breast and other cell lines and do not seem to interfere with tamoxifen while the antioxidant form of vitamin E does seem to interfere. Human absorption of vitamin E favors alpha tocopherol, and whether there are any clinical effects of these other forms of vitamin E would depend on whether there is adequate absorption. Some of our future work will include looking at the derivatives of vitamin E that would be from alphatocopherol, which are favorably taken up but which may have a nonantioxidant effect. Interestingly, we don’t know the correct physiologic levels of vitamin E. Vitamin E was identified as a deficiency in rats; at a certain level of deficiency they became unable to reproduce. The recom-
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mended daily dose for humans is just an extrapolation from the rat dose. The typical RDA for humans is 15 milligrams a day, and the normal serum levels are about 9 to 28 micromolar. The typical supplements are 400 milligrams daily and up. There have been epidemiologic studies that show that women with a serum vitamin E level over 22 micromolar may have a higher risk of death from breast cancer.
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We are interested in looking at alternative analogues of vitamin E that might have an anticancer effect; we are also looking at biomarkers of tamoxifen resistance that may be reversible in patients who are taking vitamin E. The effect of vitamin E on aromatase inhibitors is unknown. It depends on whether there is any action of the aromatase inhibitors in creating oxidative stress, for instance, by greatly reducing the estrogen in the cell.
Background. Induction of apoptosis by tamoxifen has been postulated to involve oxidative stress. Tamoxifen (TAM) may act on estrogen receptors (ER) located in the plasma membrane. Our hypothesis that supplemental antioxidant vitamin E (␣-tocopherol) acts at the plasma membrane to alter the effectiveness of tamoxifen was tested in ER-positive breast cancer cell lines, MCF-7 and T47D. Methods. Cells were treated in vitro with 20-M TAM alone and in combination with 10-M ␣-tocopherol (AT). Estrogen growth signals were quantified by immunohistochemical staining for the mitogen-activated protein kinase p-ERK. Rapid changes in intracellular calcium were detected in TAM-treated MCF-7 and T-47D cells by fluorescence microscopy of cells loaded with the calciumsensitive dye Fluo 4AM. Apoptosis was assayed by flow cytometry. Results. Proliferating cells in normal medium exhibited strong p-ERK staining. Addition of TAM abolished p-ERK staining and caused cell rounding and death. The addition of AT led to the restoration of cell proliferation and p-ERK expression even in the presence of high-dose TAM. Intracellular calcium rapidly increased in MCF-7 and T47D cells upon exposure to TAM, followed by an increase in caspase activation and eventual apoptosis. The increase in intracellular calcium was abolished by the addition of 10M AT to TAM, and pan-caspase staining decreased at 5 hours from 72% to 41%. Conclusions. These studies suggest that supplemental vitamin E decreases the inhibitory effect of TAM on the proliferation of ER⫹ breast cancer cells and eliminates the rapid rise in intracellular calcium that leads to apoptosis stimulated by TAM. The use of vitamin E acetate supplements may be inadvisable for women taking tamoxifen. (Surgery 2006;140:607-15.) From the Department of Surgery, Southern Illinois University School of Medicine, Springfield, Ill
A milestone in the adjuvant treatment of breast cancer was the demonstration of disease-free and overall survival benefit conferred by the selective estrogen receptor modulator, tamoxifen.1-3 Tamoxifen remains a commonly prescribed drug for treatment and prevention of hormone-responsive breast cancer. The mechanism of action of tamoxifen is through binding with the estrogen receptor-␣ (ER) and inhibiting the transcriptional activation of estrogen response elements in the Presented at the 63rd Annual Meeting of the Central Surgical Association, Louisville, Kentucky, March 9-11, 2006. Supported by the Illinois Department of Public Health Penny Severns Breast and Cervical Cancer Research Fund and the Breast Center at SIU Breast Cancer Research Fund. Accepted for publication July 10, 2006. Reprint requests: Elizabeth A. Peralta, MD, Southern Illinois University School of Medicine, P.O. Box 19638, Springfield, IL 62794-9638. E-mail: [email protected]. 0039-6060/$ - see front matter © 2006 Mosby, Inc. All rights reserved. doi:10.1016/j.surg.2006.07.007
genome.4 There has been increasing evidence for a role of estrogen receptors located in the plasma membrane in the signaling activity of tyrosine kinase receptors, such as the epidermal growth factor receptors EGFR, HER-1, and Her-2 neu.5 In breast cancers, an inverse correlation has been noted between the level of expression of ER and HER-2 neu and other EGFRs. Increased EGFR expression also has been implicated in the development of tamoxifen resistance.6,7 Similarly for tamoxifen, a variety of nongenomic pathways may contribute to the antitumor effect, possibly mediated by signaling events occurring at the cell surface. These signals include modulation of protein kinase C, calmodulin, TGF-, ceramide, activated JNK, p38, and mitochondrial toxicity with collapse of the transmembrane potential and caspase activation.8 The in vitro induction of apoptosis by tamoxifen has been postulated to involve oxidative stress.9 Given the possible importance of oxidative stress in the antitumor effect of tamoxifen, antioxidant vitamin supplementation may be counterproducSURGERY 607
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tive. 〈lpha-tocopherol acetate, the popular form of vitamin E supplement taken by many healthy adults and cancer patients, also is considered to be a harmless remedy for hot flashes. A prospective, blinded, placebo-controlled trial with crossover compared the benefit of 800 IU/day vitamin E versus placebo in women with vasomotor symptoms.10 The study involving 105 patients showed that vitamin E was associated with a minimal decrease in hot flashes (1 less hot flash per day than was seen with placebo) (P ⱕ .05). Megadose vitamin E differs from a placebo in that it influences the biochemical milieu in which other cancer therapies are acting. It has its most important function as a free-radical scavenger in the lipid compartment of cells and serum, where increased serum vitamin E levels correspond to decreased lipid peroxidation. It is a potent inhibitor of protein kinase C and 5-lipoxygenase.11,12 In vitro, vitamin E also inhibits smooth muscle cell proliferation, platelet aggregation, and the oxygen burst in neutrophils.13,14 The hypothesis that supplemental vitamin E (␣-tocopherol [AT]) alters the effectiveness of tamoxifen was tested using in vitro models of human breast carcinoma. One approach to demonstrating a nongenomic mechanism for tamoxifen effect is to assess the activation of plasma membrane–associated signaling. Intracellular calcium concentration [Ca 2⫹]i is a highly versatile second messenger in the cell involved in motility, growth, differentiation, and apoptosis.15 The steep concentration gradient between extracellular calcium and [Ca 2⫹]i allows small changes to trigger Ca2⫹-dependent events. Both estrogen and tamoxifen have been observed causing rapid rises in [Ca 2⫹]i in a variety of cultured cells including vascular endothelial cells, chicken granulosa cells, and breast cancer cells.16-18 In the present study, we measured rapid increases in [Ca 2⫹]i in ER⫹ cancer cell lines upon exposure to tamoxifen, and we related the timing and culture conditions to the onset of apoptosis. Phosphorylation of the mitogen-activated protein (MAP) kinases ERK-1 and ERK-2 has been shown to be stimulated by estradiol in the ER⫹ breast cancer cell line MCF-7. This p-ERK expression is associated with increased proliferation and is blocked by the ER antagonist ICI 182,780.19 In MCF-7, p-ERK expression is reduced by tamoxifen.20 Paired specimens of ER⫹ breast cancers before and after progression during tamoxifen treatment showed increased expression of activated p38 and p-ERK.21 Therefore, we have used p-ERK expression combined with cell proliferation as an indication of tamoxifen resistance.
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MATERIALS AND METHODS Cell lines. Human breast cancer cell lines MCF-7, T47D, and MDA MB 231 were obtained from the American Type Culture Collection (ATCC, Manassas, Va). The prostate cancer cell line LNCap was a kind gift from Dr Louis Premkumar. Cells were screened negative for mycoplasma and maintained in Dulbecco modified Eagle medium (DMEM) supplemented with 10% fetal bovine serum (FBS), 200 mM glutamine and penicillin. In vitro proliferation assays. MCF-7 and T47D cells were plated at 5000 cells/well in a 96-well plate and incubated overnight at 37°C and 5% CO2 in normal medium before challenge with ␣-tocopherol (AT) (10-100 M) alone and in combination with 10 M tamoxifen (TAM) in MCF-7 and T47D cells for 24 hours. MTS substrate (Cell Titre96 Aqueous Non-radioactive Cell Proliferation Assay, Promega Corp, Madison, Wis) was then added. Plates were incubated for 3 hours and spectrometry was performed to read absorbance at 490 nm. MCF-7 and T47D assays were run in triplicate and repeated in 3 separate experiments. Immunohistochemistry. T47D cells and MCF-7 cells were grown on sterile chambered slides with different doses of tamoxifen (10, 20, and 40M) and in combination with tamoxifen (40 M) and different doses of AT (10, 50, and 100M) for 24 hours. The slides were fixed in 4% methanol-free formaldehyde for 25 minutes at 4°C and then processed for immunohistochemistry using R.T.U Vectastain Universal ABC kit (Vector Laboratories, Burlingame, Calif). A mouse monoclonal pERK antibody (Santa Cruz Biotechnology, Inc., Santa Cruz, Calif) was used at 1:100 dilution. The cells were incubated overnight at 4°C. Negative controls were incubated with phosphate-buffered saline (PBS) for the same period. After washing, the slides were incubated for 30 minutes in biotinylated universal secondary antibody and stained according to the manufacturer’s protocol. For each slide, p-ERK staining was scored as 0 if no cells exhibited deep brown cytoplasmic staining, 1⫹ if 10% of cells were stained, and 2⫹ if more than 10% of cells were stained. Intracellular calcium imaging. The estrogen receptor-positive (ER⫹) breast cancer cell lines MCF-7 and T47D, and the estrogen receptor ␣-negative cell lines MDA-MB-231(breast carcinoma) and LNCap (an estrogen receptor -positive prostate carcinoma) were tested in normal culture medium, steroid-free medium, and medium containing 10 M AT. Cells were loaded with the
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Fig 1. Proliferation of breast cancer cells treated with tamoxifen and vitamin E. a, Effect of different doses of ␣-tocopherol (AT) and tamoxifen (TAM) at 24 hours incubation on proliferation of T47.D breast tumor cells; AT 10 M or 50 M alone had no effect. Tamoxifen-treated cells displayed a dose-dependent decrease in proliferation rates when compared to cells treated with media and vehicle. Combination treatment of MCF-7 (b) and T47D ER⫹ (c) breast cancer cell lines with 40uM TAM and various concentrations of AT displayed significantly higher proliferation rates compared to cells treated with TAM alone. *P ⬍ .005.
calcium ion (Ca2⫹)-sensitive dye Fluo 4AM (Molecular Probes, Eugene, Ore) and then transferred to a reading chamber with continuous flow of test medium. Fluo 4AM was excited at 488 nm, and the emitted fluorescence of single cells was filtered with a 535 ⫾ 25 nm bandpass filter and read into a computer using Scanalytics software (Scanalytics, Fairfax, Va). Changes in the intensity of fluorescence (F/Fo) were determined using fluorescence microscopy (DMIRE2; Leica, Plymouth, Minn) and plotted against time to evaluate changes in intracellular calcium levels [Ca 2⫹]i in the presence of TAM and TAM plus AT. Thapsigargin is an inhibitor of the Ca2⫹ ATPase that stores Ca2⫹ in the smooth endoplasmic reticulum. Thapsigargin treatment was used to test for an influx of extracellular Ca2⫹ through channels distinct from voltage-gated Ca2⫹ channels, a process termed capacitative Ca2⫹ entry.22 Apoptosis assays. MCF-7 cells were pretreated for 1 hour with 0 or 10M AT in normal medium before the addition of 20 M TAM. Apoptosis was quantified at 0, 2, and 5 hours using fluorescence-activated cell sorting and staining with FITC-VD-FMK (ApoStat; R&D Systems, Minneapolis, Minn), an indicator of pan-caspase activity. RESULTS Alpha tocopherol increases the proliferation rate of TAM-treated cells. TAM-treated T47D cells displayed a dose-dependent decrease in proliferation rate compared with cells treated with media or vehicle, with a 95% inhibition at 40 M TAM (Fig 1, a). Addition of AT to media containing 40 M TAM restored the proliferation rate to 60% of media control (Fig 1, b). In MCF-7 cells, 40 M TAM
Fig 2. Effect of AT on cell number and morphology in TAM-treated cells. a, T47D cells cultured on chambered slides in 40 M TAM alone and with 50 M AT. b, MCF7 cells cultured on chambered slides in 40 M TAM alone and with 50 M AT (Hematoxylin/eosin stain; magnification: X200).
reduced proliferation by 68%. Addition of AT to 40 M TAM restored the proliferation rate from 32% to 100% of media control (Fig 1, c). The effect was significant at all concentrations of AT, but was maximal in the range of 10 to 100 M. Culturing cells under these media conditions on chambered slides and staining with hematoxylin/eosin shows scarce, pyknotic cells with TAM treatment compared with confluent, viable cells exposed to TAM⫹AT (Fig 2, a and b). These findings demon-
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Fig 3. Effect of AT on p-ERK expression in TAM-treated cells. a, MCF-7 cells negative control; b, MCF-7 cells in normal medium (positive control); c, MCF-7 cells treated for 24 hours with Tam 40 M; d, MCF-7 cells treated with combination of Tam 40 M and AT 50 M. e, T47D cells negative control; f, T47D cells in normal medium (positive control); g, T47D cells treated for 24 hours with Tam 40 M; h, T47D cells treated with combination of Tam 40 M and AT 50 M. (p-ERK immunostain; magnification: X200).
strate that AT can cause tamoxifen resistance in hormone-sensitive cells. P-ERK expression is restored in TAM-treated cells exposed to alpha-tocopherol. MCF-7 and T47D both express p-ERK when growing in normal medium that contains estrogen from fetal calf serum (Fig 3, b and f). P-ERK expression was decreased to 0-1⫹ in TAM-treated cells (Fig 3, c and g). Addition of AT to TAM restored expression of p-ERK to 2⫹ and increased the number and viability of both MCF7 and T47D cells (Fig 3, d and h). From these experiments, it is postulated that p-ERK expression is associated with TAM-resistance in hormone-sensitive breast cancer cells. Alpha tocopherol prevents the increase of intracellular calcium, a signal preceding apoptosis of ERⴙ tumor cells. Exposure to TAM (5 to 20M) stimulated a rapid (onset at 4 minutes) and significant increase in [Ca 2⫹]i in T47D and MCF-7 cells (Fig 4, a and b). The signal was self-limiting, reproducible, and was stronger with greater TAM dose (Fig 4, c). A striking finding was that the increase in [Ca 2⫹]i was abolished by flowing medium containing AT over the cells for 15 minutes between TAM exposures and by preincubation with AT for 24 hours prior to TAM treatment (Fig 4, d and e). Removing Ca2⫹ from the bathing medium abolished the rise in [Ca 2⫹]i. With Ca2⫹ removed from the extra cellular medium, intracellular Ca2⫹ stores are not released by TAM, as demonstrated by the transient rise in [Ca 2⫹]i when thapsigargin is added after TAM (Fig 4, f). These experiments indicate that the increase in concentration of intracellular calcium in MCF-7 and T47D cells is not by release of Ca2⫹
from endoplasmic reticulum stores and requires Ca2⫹entry from the extracellular medium. ER expression was necessary for TAM to elicit the [Ca 2⫹]i signal. TAM did not stimulate an increase in [Ca 2⫹]i in the estrogen receptor ␣-negative cell lines MDA-MB-231, LNCap, or MCF-7 cells that had been incubated in steroid-free medium to reduce ER expression (Fig 4, g-i). These results support a link between the absence of TAM-stimulated intracellular calcium flux and tamoxifen-resistance. Alpha-tocopherol decreases tamoxifen-induced apoptosis in vitro in ERⴙ breast cancer cell lines. MCF-7 cells are defective for caspase-3, a rapid effector caspase, but MCF-7 cells can undergo activation of other caspases and morphologic changes of apoptosis with TAM treatment with an onset of 90 minutes.23 We used a pan-caspase fluorescent substrate to quantify apoptosis by fluorescenceactivated cell sorting. The population of apoptotic MCF-7 cells at 5 hours was 72% in cells treated with 20 M TAM alone (Fig 5, a) compared with 41% in cells treated with TAM plus 10 M AT (Fig 5, b). DISCUSSION Cancer patients frequently report use of vitamin E supplements during and after therapy. A casecontrol study in which interviews with Wisconsin women aged 18 to 69 years who were recent breast cancer survivors were compared with interviews of randomly selected controls found that cancer survivors were 5.3 times more likely than controls to experience menopausal symptoms and 7.4 times more likely to use alternative remedies such as soy, vitamin E, or herbs.24 Because the mechanism of
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Fig 4. Effect of tamoxifen on intracellular calcium (Cai). a, T47.D Breast cancer cells; b, MCF-7 breast cancer cells. c, Effect of different doses of tamoxifen 5-M and 20-M doses on T4.7 cells. Bar indicates the time of Tam addition. Individual cell tracings recorded simultaneously are shown. d, The increase in Cai after exposure to tamoxifen 20 uM was abolished by 10 M AT in T47.D cells by flowing ␣-tocopherol acetate between Tamoxifen exposures. e, In MCF-7 cells, preincubation with AT for 24 hours abolished the rise in Cai when Tam is added. f, Effect of removing extracellular Ca2⫹ on intracellular calcium stores (a) 1 uM Thapsigargin and 20 um Tamoxifen were added as indicated. After releasing smooth endoplasmic reticulum calcium stores with thapsigargin, administration of Tamoxifen did not cause an increase in Cai. g, Effect of Tam dose on Cai in ER-negative MDA-MB-231 cells; h, same for ER ␣-negative, ER -positive LNCap cells (prostate); i, same for MCF-7 cells cultured in steroid-free medium to decrease ER expression. Tamoxifen displayed minimal or no effect in these cells.
action of certain therapies such as radiation and chemotherapy involve the generation of oxygen radicals, it is a matter of concern whether antioxidant supplements should be used. Although the magnitude of the in vitro effect of AT in this study was large, it may not necessarily
reflect the in vivo impact of dietary AT. We supplemented the cell culture medium with vitamin E to concentrations of 10 to 50 M. The normal human serum level of vitamin E ranges from 11.6 to 38 M and is dependent on serum lipid levels and activity of AT transfer protein in the liver. The recom-
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Fig 5. Vitamin E reduces tamoxifen-induced apoptosis in vitro. MCF-7 cells were pretreated for 1 hour with 0 (a) or 10 (b) M AT before the addition of 20 M TAM. Apoptosis is shown at 5 hours using flow cytometry and staining with FITC-VD-FMK (R&D Systems), an indicator of pan-caspase activity.
mended daily allowance of vitamin E is 15 mg/day compared to the average over-the-counter vitamin E capsule, which contains 400 mg. When supplements are taken, the serum level does not predictably rise. A study of volunteers given a dose of 75 mg natural AT resulted in a plasma concentration rise at 12 hours from 0.3 to 12.4 M.25 The Nurses Health Study, a cohort of 83,234 women enrolled in 1976, has been followed by Zhang et al for multivariate analyses of breast cancer risk and lifestyle factors including dietary intakes of carotenoids and vitamins A, C, and E.26 Whereas consumption of fruits and vegetables high in specific carotenoids was associated with a mild reduction in breast cancer risk in premenopausal women, total vitamin E from food and supplements was related to a weak increased risk of premenopausal breast cancer. Zhang and colleagues speculated that perhaps women with worse fibrocystic breast disease might have been more likely to take vitamin E supplements. Given that vitamin E is not contributory to breast cancer prevention and may antagonize the effect of tamoxifen, it seems inadvisable for women who take tamoxifen for chemprevention of breast cancer also take vitamin E supplements. An important clinical use of tamoxifen is in the adjuvant setting, ie, in women with no evidence of disease. In such a setting, it is difficult to assess the effect of vitamin E on tamoxifen unless a difference in relapse rate can be observed. In a prospective study of 363 patients newly diagnosed with breast cancer, blood was collected prior to therapy to determine levels of vitamin E, plasma cholesterol, lipoperoxides, and glutathione oxidase. There was an inverse relationship between tumor size and plasma lipoperoxides, and higher lipoperoxide levels were associated with lower-stage (node-negative) disease. A total of 66% of the patients had ER⫹ tumors and 89% of these received adjuvant hormonal therapy. With a median follow-up of 8 years, they found an increased risk of
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recurrence ([RR] 1.7) and breast cancer death (RR 2.0) in patients with plasma-lipid adjusted vitamin E levels above 22 mol/L.27 Other studies have found that higher vitamin E intake correlated with increased treatment failure within 2 years in women with ER⫹ breast cancer.28 An interaction between tamoxifen and vitamin E in breast cancer cell lines has been previously observed. Tamoxifen caused growth inhibition via downregulation of protein kinase C in ER-negative MDA MB 231 cells.9 In that experiment, TAM was found to partition in the plasma membrane, and the growth inhibition was abolished by antioxidants such as vitamin E and superoxide dismutase. In other ER-negative cells, TAM induced apoptosis through JNK1 and caspase-3 activation. Apoptosis was blocked by vitamin E but not the water-soluble antioxidants N-acetyl L-cysteine and glutathione.29 In contrast, nonantioxidant vitamin E analogues such as the tocotrienols and ␣-tocopherol succinate (␣-TOS) have a proapoptotic effect on breast and other cancer cell lines.30-32 Multiple signaling pathways have been implicated including activation of the caspase 3 cascade, inhibition of PKC␣, and activation of JNK1.33 In animal models, ␣-TOS had to be administered by intraperitoneal injection; oral administration leads to hydrolysis of ␣-TOS to AT, which had no antitumor effect. These observations of nonantioxidant vitamin E analogues are not inconsistent with our hypothesis that the antioxidant effect of vitamin E in the plasma membrane may interfere with tamoxifen-induced apoptosis. The present findings suggest that the mechanism for vitamin E–induced tamoxifen resistance involves interruption of plasma membrane ER signaling via blocking a Ca2⫹ second messenger and increasing p-ERK activation. Recently, an estrogen receptor of a different class, a G protein-coupled receptor (GPR30), has been described.34 GPR30 may have a crucial role in the estrogenstimulated activation of p-ERK. It was observed that 17beta-estradiol activates ERK-1/-2 in MCF-7 cells, which express both estrogen receptor alpha (ER alpha) and ER beta, and in SKBR3 breast cancer cells, which fail to express either receptor. Immunoblot analysis using GPR30 peptide antibodies showed that this estrogen response was associated with the presence of GPR30 protein in these cells.35 GPR30 binds not only 17beta-estradiol but also 1tamoxifen and ICI 182,780 with an agonist signal mimicking estrogen.36 This G-protein activation results in the activation of cascades of signal transduction including mobilization of intracellular calcium, production of cAMP, stimulation of phosphlipase C, protein kinase C, and other proteins. It
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is unknown how vitamin E may affect GPR30 signaling. A hypothesis that fits our findings to date is that vitamin E may block the proapoptotic Ca2⫹ signal while TAM binding to GPR30 activates p-ERK, thus giving a net growth-stimulatory effect of TAM at the cell membrane and contributing to tamoxifen-resistance. The efficacy of tamoxifen as it is used in practice, ie, in combination with vitamin supplements, might be assessable with surrogate endpoint biomarkers. Candidate biomarkers are Ki67, ER, PR, and p-ERK in ductal epithelial cells from normal breast biopsies of patients in adjuvant therapy. We are currently conducting a pilot study of these biomarkers in women who are already taking vitamin E concurrent with tamoxifen and are willing to undergo random core breast biopsies 4 weeks before and after they discontinue vitamin E. Such a study may provide insight into the interaction of oxidative status and selective estrogen receptor modulation and identify useful biomarkers to assess other prevention drugs. In the meanwhile, it appears prudent to discourage vitamin E supplementation in women taking tamoxifen. The authors thank Dr. Sophia Ran for editorial assistance.
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REFERENCES 1. Fisher B, Redmond C, Legault-Poisson S, Dimitrov NV, Brown AM, Wickerham DL, et al. Postoperative chemotherapy and tamoxifen compared with tamoxifen alone in the treatment of positive-node breast cancer patients aged 50 years and older with tumors responsive to tamoxifen: results from the National Surgical Adjuvant Breast and Bowel Project B-16. J Clin Oncol 1990;8:1005-18. 2. Fisher B, Dignam J, Bryant J, DeCillis A, Wickerham DL, Wolmark N, et al. Five versus more than five years of tamoxifen therapy for breast cancer patients with negative lymph nodes and estrogen receptor-positive tumors. J Natl Cancer Inst 1996;88:1529-42. 3. Early Breast Cancer Trialists’ Collaborative Group. Effects of adjuvant tamoxifen and of cytotoxic therapy on mortality in early breast cancer. An overview of 61 randomized trials among 28,896 women. N Engl J Med 1988;319:1681-92. 4. MacGregor JI, Jordan VC. Basic guide to the mechanisms of antiestrogen action. Pharmacol Rev 1998;50:151-96. 5. Schiff R, Massarweh SA, Shou J, Bharwani L, Mohsin SK, Osborne CK. Cross-talk between estrogen receptor and growth factor pathways as a molecular target for overcoming endocrine resistance. Clin Cancer Res 2004;10:331S-6S. 6. Gee JM, Robertson JF, Ellis IO, Nicholson RI. Phosphorylation of ERK1/2 mitogen-activated protein kinase is associated with poor response to anti-hormonal therapy and decreased patient survival in clinical breast cancer. Int J Cancer 2001;95:247-54. 7. Shou J, Massarweh S, Osborne CK, Wakeling AE, Ali S, Weiss H, et al. Mechanisms of tamoxifen resistance: in-
17.
18.
19.
20.
21.
22.
23.
creased estrogen receptor-HER2/neu cross-talk in ER/HER2-positive breast cancer. J Natl Cancer Inst 2004;96:926-35. Mandlekar S, Kong AN. Mechanisms of tamoxifen-induced apoptosis. Apoptosis 2001;6:469-77. Gundimeda U, Chen ZH, Gopalakrishna R. Tamoxifen modulates protein kinase C via oxidative stress in estrogen receptor-negative breast cancer cells. J Biol Chem 1996;271: 13504-14. Barton DL, Loprinzi CL, Quella SK, Sloan JA, Veeder MH, Egner JR, et al. Prospective evaluation of vitamin E for hot flashes in breast cancer survivors. J Clin Oncol 1998;16:495-500. Ricciarelli R, Zingg JM, Azzi A. Vitamin E: protective role of a Janus molecule. Faseb J 2001;15:2314-25. Pratico D, Tangirala RK, Rader DJ, Rokach J, FitzGerald GA. Vitamin E suppresses isoprostane generation in vivo and reduces atherosclerosis in ApoE-deficient mice. Nat Med 1998;4:1189-92. Brigelius-Flohe R, Kelly FJ, Salonen JT, Neuzil J, Zingg JM, Azzi A. The European perspective on vitamin E: current knowledge and future research. Am J Clin Nutr 2002;76:703-16. Wesselborg S, Bauer MK, Vogt M, Schmitz ML, SchulzeOsthoff K, et al. Activation of transcription factor NFkappaB and p38 mitogen-activated protein kinase is mediated by distinct and separate stress effector pathways. J Biol Chem 1997;272:12422-9. Krebs J. The role of calcium in apoptosis. Biometals 1998; 11:375-82. Rubio-Gayosso I, Sierra-Ramirez A, Garcia-Vazquez A, Martinez-Martinez A, Munoz-Garcia O, Morato T, et al. 17Betaestradiol increases intracellular calcium concentration through a short-term and nongenomic mechanism in rat vascular endothelium in culture. J Cardiovasc Pharmacol 2000; 36:196-202. Morley P, Whitfield JF, Vanderhyden BC, Tsang BK, Schwartz JL. A new, nongenomic estrogen action: the rapid release of intracellular calcium. Endocrinology 1992;131: 1305-12. Chang HT, Huang JK, Wang JL, Cheng JS, Lee KC, Lo YK, et al. Tamoxifen-induced increases in cytoplasmic free Ca2⫹ levels in human breast cancer cells. Breast Cancer Res Treat 2002;71:125-31. Migliaccio A, Di Domenico M, Castoria G, de Falco A, Bontempo P, Nola E, et al. Tyrosine kinase/p21ras/MAPkinase pathway activation by estradiol-receptor complex in MCF-7 cells. Embo J 1996;15:1292-300. Yang D, Wang S, Ye Y, Cui Z. [Effect of mitogen-activated protein kinase signal transduction in non-estrogen antagonistic mechanism of tamoxifen]. Zhonghua Yi Xue Za Zhi 2002;82:1232-4. Gutierrez MC, Detre S, Johnston S, Mohsin SK, Shou J, Allred DC, et al. Molecular changes in tamoxifen-resistant breast cancer: relationship between estrogen receptor, HER-2, and p38 mitogen-activated protein kinase. J Clin Oncol 2005;23:2469-76. Hunton DL, Lucchesi PA, Pang Y, Cheng X, Dell’Italia LJ, Marchase RB. Capacitative calcium entry contributes to nuclear factor of activated T-cells nuclear translocation and hypertrophy in cardiomyocytes. J Biol Chem 2002;277: 14266-73. Rehm M, Dussmann H, Janicke RU, Tavare JM, Kogel D, Prehn JH. Single-cell fluorescence resonance energy transfer analysis demonstrates that caspase activation during ap-
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24.
25.
26.
27.
28.
29.
30.
31. 32.
33.
34.
35.
36.
optosis is a rapid process. Role of caspase-3. J Biol Chem 2002;277:24506-14. Harris PF, Remington PL, Trentham-Dietz A, Allen CI, Newcomb PA. Prevalence and treatment of menopausal symptoms among breast cancer survivors. J Pain Symptom Manage 2002;23:501-9. Roxborough HE, Burton GW, Kelly FJ. Inter- and intraindividual variation in plasma and red blood cell vitamin E after supplementation. Free Radic Res 2000;33:437-45. Zhang S, Hunter DJ, Forman MR, Rosner BA, Speizer FE, Colditz GA, et al. Dietary carotenoids and vitamins A, C, and E and risk of breast cancer. J Natl Cancer Inst 1999;91:547-56. Saintot M, Mathieu-Daude H, Astre C, Grenier J, SimonyLafontaine J, Gerber M. Oxidant-antioxidant status in relation to survival among breast cancer patients. Int J Cancer 2002;97:574-9. Holm LE, Nordevang E, Hjalmar ML, Lidbrink E, Callmer E, Nilsson B. Treatment failure and dietary habits in women with breast cancer. J Natl Cancer Inst 1993;85:32-6. Mandlekar S, Yu R, Tan TH, Kong AN. Activation of caspase-3 and c-Jun NH2-terminal kinase-1 signaling pathways in tamoxifen-induced apoptosis of human breast cancer cells. Cancer Res 2000;60:5995-6000. Neuzil J, Weber T, Schroder A, Lu M, Ostermann G, Gellert N, et al. Induction of cancer cell apoptosis by alpha-tocopheryl succinate: molecular pathways and structural requirements. Faseb J 2001;15:403-15. Malafa MP, Neitzel LT. Vitamin E succinate promotes breast cancer tumor dormancy. J Surg Res 2000;93:163-70. Guthrie N, Gapor A, Chambers AF, Carroll KK. Inhibition of proliferation of estrogen receptor-negative MDA-MB-435 and -positive MCF-7 human breast cancer cells by palm oil tocotrienols and tamoxifen, alone and in combination. J Nutr 1997;127:544S-8S. Yu W, Simmons-Menchaca M, You H, Brown P, Birrer MJ, Sanders BG, et al. RRR-alpha-tocopheryl succinate induction of prolonged activation of c-jun amino-terminal kinase and c-jun during induction of apoptosis in human MDA-MB-435 breast cancer cells. Mol Carcinog 1998;22:247-57. Carmeci C, Thompson DA, Ring HZ, Francke U, Weigel RJ. Identification of a gene (GPR30) with homology to the G-protein-coupled receptor superfamily associated with estrogen receptor expression in breast cancer. Genomics 1997;45:607-17. Filardo EJ, Quinn JA, Bland KI, Frackelton AR Jr. Estrogen-induced activation of Erk-1 and Erk-2 requires the G protein-coupled receptor homolog, GPR30, and occurs via trans-activation of the epidermal growth factor receptor through release of HB-EGF. Mol Endocrinol 2000;14: 1649-60. Thomas P, Pang Y, Filardo EJ, Dong J. Identity of an estrogen membrane receptor coupled to a G-protein in human breast cancer cells. Endocrinology 2005;146:624-32.
DISCUSSION Dr Aness Chagpar (Louisville, Ky): In this study, Dr Peralta and her colleagues from the Southern Illinois University School of Medicine investigate the effect of combining alpha-tocopherol acetate, a component of vitamin E, with tamoxifen on breast cancer cell line growth, apoptosis and intracellular signaling.
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In an era of growing interest in alternative and complementary therapies, their work is particularly timely. They demonstrated that the addition of alpha-tocopherol decreased the inhibitory effects of tamoxifen on the proliferation of estrogen receptor positive breast cancer cells, and, furthermore, that it eliminated the rapid rise in intracellular calcium leading to apoptosis. A number of investigators have found that alphatocopherol may blunt the inhibitory effect of tamoxifen. Tocotrienols, however, which are another component of vitamin E formulation, have not been shown to have the same effect in some studies. Did you look at tocotrienols and could you comment on what could account for the difference between the tocotrienols and alpha-tocopherol? What is the dose of vitamin E that we get with oral supplementation and how does that tie into your results? There is newer data suggesting that aromatase inhibitors, which act through a different mechanism through peripheral conversion, may be more efficacious than tamoxifen in postmenopausal women. Given these data, could you speculate on what effect you might expect with vitamin E derivatives and their effect on aromatase inhibitors? Dr Gerald M. Larson (Louisville, Ky): Could you tell us how large the subset of patients is that takes both vitamin E and tamoxifen? Dr Elizabeth A. Peralta: The subset of people we are interested in are women who are taking tamoxifen either for breast cancer adjuvant therapy or prevention. In my practice following up with patients, I would ask them what they are taking in addition to their prescription medications, and I found that vitamin E was rather common. What I have learned in looking at some of the other components of vitamin E is that the nonantioxidant forms of vitamin E such as the tocotrienols have an antitumor or proapoptotic effect on breast and other cell lines and do not seem to interfere with tamoxifen while the antioxidant form of vitamin E does seem to interfere. Human absorption of vitamin E favors alpha tocopherol, and whether there are any clinical effects of these other forms of vitamin E would depend on whether there is adequate absorption. Some of our future work will include looking at the derivatives of vitamin E that would be from alphatocopherol, which are favorably taken up but which may have a nonantioxidant effect. Interestingly, we don’t know the correct physiologic levels of vitamin E. Vitamin E was identified as a deficiency in rats; at a certain level of deficiency they became unable to reproduce. The recom-
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mended daily dose for humans is just an extrapolation from the rat dose. The typical RDA for humans is 15 milligrams a day, and the normal serum levels are about 9 to 28 micromolar. The typical supplements are 400 milligrams daily and up. There have been epidemiologic studies that show that women with a serum vitamin E level over 22 micromolar may have a higher risk of death from breast cancer.
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We are interested in looking at alternative analogues of vitamin E that might have an anticancer effect; we are also looking at biomarkers of tamoxifen resistance that may be reversible in patients who are taking vitamin E. The effect of vitamin E on aromatase inhibitors is unknown. It depends on whether there is any action of the aromatase inhibitors in creating oxidative stress, for instance, by greatly reducing the estrogen in the cell.