- Email: [email protected]
Pterostilbene and tamoxifen show an additive effect against breast cancer in vitro
The American Journal of Surgery (2010) 200, 577–580
Association of VA Surgeons
Pterostilbene and tamoxifen show an additive effect against breast cancer in vitro Patrick Mannal, M.D.*, Debbie McDonald, B.S., David McFadden, M.D., M.B.A., F.A.C.S. Division of Surgical Oncology, The University of Vermont/Fletcher Allen Health Care, Burlington, VT, USA KEYWORDS: Pterostilbene; Tamoxifen; Breast cancer; Apoptosis
Abstract BACKGROUND: Tamoxifen is widely used for the treatment of breast cancer. Pterostilbene, a bioavailable stilbenoid found in blueberries, has been found to inhibit breast cancer growth in vitro. It was hypothesized that combining pterostilbene with tamoxifen would produce additive effects on estrogen receptor–positive breast cancer cells. METHODS: Two estrogen receptor–positive breast cancer cell lines, MCF7 and ZR-751, were pretreated with graduated doses of pterostilbene for 24 hours, followed by 5 mol/L tamoxifen. MTT proliferation assays and Cell Death Detection ELISAPLUS tests evaluated cell viability and apoptosis. RESULTS: MCF7 cells showed inhibition (10 and 20 mol/L, P ⬍ .001; 30 mol/L, P ⬍ .05) at all time points when combined with tamoxifen. ZR-751 cells showed additive reductions in cell viability (P ⬍ .001). Cell Death Detection ELISAPLUS indicated increased apoptosis (P ⬍ .01). CONCLUSIONS: Pterostilbene shows an additive inhibitory effect on breast cancer cells when combined with tamoxifen, most likely from augmented cancer cell apoptosis. © 2010 Elsevier Inc. All rights reserved.
Breast cancer is the second leading cause of cancer mortality in women,1 with 191,000 new cases this year.2 Major advances have been in the area of adjuvant therapy, specifically selective estrogen receptor (ER) modulators and aromatase inhibitors.3,4 Epidemiologic studies suggest that a diet rich in fruits and vegetables is associated with a reduced risk for a number of common cancers. Phytochemicals are found in plants and have protective or preventive properties. Stilbenes are 1 class of phytochemicals shown to have antioxidant and antiproliferative properties. Resveratrol, a stilbene found in grapes, has been shown to inhibit a variety of primary tumors.5–9 Pterostilbene, an analogue of resveratrol found in blueberries, has been shown to inhibit tumor growth in various models10 –15 and is more bioavailable than * Corresponding author: Tel.: 802-656-3319; fax: 802-656-0680. E-mail address: [email protected] Manuscript received May 18, 2010; revised manuscript July 7, 2010
0002-9610/$ - see front matter © 2010 Elsevier Inc. All rights reserved. doi:10.1016/j.amjsurg.2010.07.022
resveratrol upon oral ingestion. We hypothesized that combining pterostilbene with tamoxifen would produce an additive effect on ER-positive breast cancer in vitro. In addition, we evaluated the aromatase inhibitor anastrozole because of its increasing role as an adjuvant therapy.
Methods Pterostilbene (3,5-dimethoxy-4-hydroxystilbene), 4-hydroxytamoxifen (trans-4-[1-(4-[2-[dimethylamino]ethoxy] phenyl)-2-phenyl-1-butenyl] phenol), anastrozole (2-[3-[1cyano-1-methyl-ethyl]-5-[1H-1,2,4-triazol-1-ylmethyl] phenyl]-2methyl-propanenitrile), and MTT, a tetrazolium dye (thiazolyl blue tetrazolium bromide), were purchased from Sigma-Aldrich (St Louis, MO). Cell Death Detection ELISAPLUS was purchased from Roche (Mannhiem, Germany).
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The American Journal of Surgery, Vol 200, No 5, November 2010
Results Pterostilbene and tamoxifen MTT assay, combined treatments
Figure 1 MCF-7 cells: pterostilbene pretreatment followed by tamoxifen treatment. Combined results were statistically significant (10 mol/L pterostilbene plus 5 mol/L tamoxifen, P ⱕ .001; 20 mol/L pterostilbene plus 5 mol/L tamoxifen, P ⱕ .05; 30 mol/L pterostilbene plus 5 mol/L tamoxifen, P ⫽ NS).
Breast cancer lines (MCF-7, ZR-75-1, and MDA-MB-231) were purchased from American Type Culture Collection (Manassas, VA). For experiments, cells were harvested from culture monolayers at 80% to 90% confluency. ER-positive MCF-7 and ZR-75-1 cells, as well as ERnegative MDA-MB-231 cells, were added to 96-well plates at 104 cells/well and incubated for 24 hours for adherence. After 24 hours, the cells were pretreated with pterostilbene at 10, 20, and 30 mol/L concentrations for 24 hours. After 24 hours of pterostilbene pretreatment, tamoxifen was added to the culture medium for a final concentration of 5 mol/L tamoxifen. Conversely, the MCF-7 and ZR-75-1 cell lines were pretreated with 5 mol/L tamoxifen for 24 hours, followed by the addition of 10, 20, or 30 mol/L pterostilbene. In addition, MCF-7, ZR-75-1, and MDA-MB231 cells were pretreated for 24 hours with 10, 20, and 30 mol/L pterostilbene, followed by 24, 48, and 72 hours of 50 mol/L anastrozole. Similarly, all cell lines were pretreated with 50 mol/L anastrozole for 24 hours, followed by 24, 48, and 72 hours of 10, 20, and 30 mol/L of pterostilbene. Control cells were treated with Dimethyl sulfoxide (DMSO). After 24, 48, and 72 hours, viability was assessed using MTT assay. Cell death pathways were analyzed using the Cell Death Detection ELISAPLUS protocol at 24 hours. Cells were treated with 20 mol/L pterostilbene for 18 hours, 5 mol/L tamoxifen for 18 hours, or the combination of 20 mol/L pterostilbene for 6 hours followed by 5 mol/L tamoxifen for an additional 18 hours. Samples were assessed for necrosis and apoptosis. Statistical comparisons among groups were performed using Student’s t test or analysis of variance, followed by Bonferroni’s posttests for multiple comparisons.
MCF-7 cells had reductions in cell viability with combined treatment of pterostilbene and tamoxifen. With pterostilbene pretreatment, the effect at 24 hours was 39%, 33%, and 31% (P ⬍ .001) of control for 10, 20, and 30 mol/L pterostilbene, respectively. Cells in the 24-hour group, treated with only 5 mol/L tamoxifen, had 58% cell viability. For the 48-hour tamoxifen treatment group, 26%, 24% (P ⬍ .001), and 24% of cells were viable after the combination of 10, 20, and 30 mol/L pterostilbene and 5 mol/L tamoxifen, with cells receiving 5 mol/L tamoxifen having 42% viability. The 72-hour tamoxifen group had 23% (P ⬍ .001), 23% (P ⱕ .05), and 23% viability with 10, 20, and 30 mol/L pterostilbene. Thirty percent of cells treated only with tamoxifen were viable at 72 hours (Fig. 1). With tamoxifen as the pretreatment agent, there were also reductions in cell viability. Cells treated with 10, 20, and 30 mol/L pterostilbene for 24 hours, after tamoxifen, had 50%, 40%, and 37% (P ⬍ .001) viability, respectively. At 48 hours, the same concentrations of pterostilbene produced 29%, 26%, and 25% (P ⬍ .001) viability. The 72hour pterostilbene group had 25%, 24% (P ⬍ .001), and 24% cell viability after tamoxifen pretreatment (Fig. 2). As with the MCF7 cells, ZR-75-1 cells had reductions in number after combined treatment. With pterostilbene pretreatment of 10, 20, and 30 mol/L for 24 hours, followed by 24 hours of 5 mol/L tamoxifen, the cells had 64%, 61%, and 42% (P ⬍ .001) viability, respectively. At 48
Figure 2 MCF-7 cells: tamoxifen pretreatment followed by pterostilbene treatment. Combined results were statistically significant (5 mol/L tamoxifen plus 10 mol/L pterostilbene, P ⱕ .001; 5 mol/L tamoxifen plus 20 mol/L pterostilbene, P ⱕ .001; 5 mol/L tamoxifen plus 30 mol/L pterostilbene, P ⫽ NS).
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Pterostilbene helps tamoxifen fight breast cancer
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combination treatment resulted in a .4-fold increase in apoptosis (value ⫽ 1.4). Treatment with 20 mol/L pterostilbene resulted in a .4-fold increase in apoptosis (value ⫽ 1.4), while 5 mol/L tamoxifen resulted in a .1-fold increase in apoptosis (value ⫽ 1.1). The combination of agents resulted in a value of 2.6, or a 1.6-fold increase over controls. Regardless of the treatment, the MDA-MB-231 cells did not display any increase in apoptosis, with all 3 categories (20 mol/L pterostilbene, 5 mol/L tamoxifen, and the combination of pterostilbene and tamoxifen) resulting in a value of .7.
Comments Figure 3 ZR-75-1 cells: pterostilbene pretreatment followed by tamoxifen treatment. All combined results were statistically significant (P ⱕ .001).
hours, viability was 51%, 36%, and 22% (P ⬍ .001). At 72 hours, 35%, 14%, and 5% (P ⬍ .001) of cells were viable after tamoxifen (Fig. 3). With 24 hours of tamoxifen pretreatment, the ZR-75-1 cells produced similar results (Fig. 4). The ER-negative cell line, MDA-MB-231, was exposed to pterostilbene pretreatments of 20 and 30 mol/L for 24 hours, followed by 5 mol/L tamoxifen for 24, 48, and 72 hours. Essentially no additive effects were seen, although pterostilbene did inhibit cell growth alone (P ⬍ .01).
Pterostilbene and anastrozole MTT assay, combined treatment
Previous studies have clearly shown the antineoplastic effects of pterostilben.16 We chose to examine the potential additive effects pterostilbene and tamoxifen, a widely used selective ER modulator, in the treatment of ER-positive breast cancer. We also evaluated the combination of anastrozole and pterostilbene. The results of the ER-positive cell line viability assays indicate that the combination of tamoxifen and pterostilbene is additive. At low doses and short time intervals, the 2 agents clearly show a greater effect together than either alone. Our inclusion of the ER-negative MDA-MB-231 cell line helped rule out a bystander effect of the tamoxifen on the target cells. Unfortunately, the mechanism of action wherein pterostilbene acts on target cells is incompletely understood, so an explanation for this effect would be speculative at best. In support of other studies examining the effect pterostilbene on other cancers, the results of our Cell Death Detection ELISAPLUS assays seem to validate an augmented
MCF-7 cells showed no additive reduction in cell viability at any time points or concentrations with combination therapy. In ZR-75-1 cells, 50 mol/L anastrozole pretreatment (24 hours) followed by the increasing doses of pterostilbene did produce significance. At 24 hours, the 10 mol/L pterostilbene treatment group had 80% cell viability (P ⬍ .01), and the 20 mol/L group had 68% viability (P ⬍ .001). After 48 hours of pterostilbene, all 3 groups had reductions to 68%, 55%, and 47% (P ⬍ .001) in response to the 10, 20, and 30 mol/L treatments, respectively. At 72 hours, the 10 mol/L pterostilbene group wase at 61% (P ⬍ .001), and the 20 mol/L group was at 41% (P ⬍ .01).
Pterostilbene and tamoxifen, Cell Death Detection ELISAPLUS Results of the Cell Death Detection ELISAPLUS are provided in fold increases of apoptosis. The MCF-7 cells, using DMSO-treated-only cells as a control, had a .2-fold increase (value ⫽ 1.2) in apoptosis with 20 mol/L pterostilbene treatment. With 5 mol/L tamoxifen treatment, MCF-7 demonstrated no increase in apoptosis (value ⫽ 1), and with
Figure 4 ZR-75-1 cells: tamoxifen pretreatment followed by pterostilbene treatment. All combined results were statistically significant (P ⱕ .001).
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The American Journal of Surgery, Vol 200, No 5, November 2010
apoptotic effect seen in the target cells. There was an obvious increase in apoptosis in the ER-positive cells with no effect on the ER-negative cells. Again, the increase in ERpositive activity was not surprising given the known mechanism of action of tamoxifen. Pterostilbene has been shown to be nontoxic in normal tissues, and our current doses do not exceed physiologic levels of ingestion. Its ability to augment the effects of conventional chemotherapy presents several potential opportunities for its use. First, lower dose conventional therapy may be possible while maintaining its antitumoral effects. This would result in lower costs and an improved side-effect profile. Second, additional antitumoral effects may be seen with conventional doses, thereby extending disease-free survival. In conclusion, the in vitro addition of pterostilbene to tamoxifen resulted in additive antiproliferative effects. Further studies are warranted.
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References 1. Altekruse SF, Kosary CL, Krapcho M, et al, eds. SEER cancer statistics review, 1975–2007, National Cancer Institute. Available at: http:// seer.cancer.gov/csr/1975_2007/. 2. US Cancer Statistics Working Group. United States cancer statistics: 1999 –2006 incidence and mortality Web-based report. Available at: http://www.cdc.gov/uscs. 3. Early Breast Cancer Trialists’ Collaborative Group. Effects of chemotherapy and hormonal therapy for early breast cancer on recurrence and 15 year survival on overview of the randomised trials. Lancet 2005;365:1687–717. 4. Osborne CK. Tamoxifen in the treatment of breast cancer. N Engl J Med 1998;339:1609 –18. 5. Lee MH, Choi BY, Kundu JK, et al. Resveratrol suppresses growth of human ovarian cancer cells in culture and in a murine xenograft model:
13.
14.
15.
16.
eukaryotic elongation factor 1A2 as a potential target. Cancer Res 2009;69:7449 –58. Harper CE, Cook LM, Patel BB, et al. Genistein and resveratrol, alone and in combination, suppress prostate cancer in SV-40 tag rats. Prostate 2009;69:1668 – 82. Chen J, Dong XS, Guo XG. Inhibitory effect of resveratrol on the growth of human colon cancer ls174t cells and its subcutaneously transplanted tumor in nude mice and the mechanism of action. Zhonghua Zhong Liu Za Zhi 2009;1:15–9. Weng CJ, Yang YT, Ho CT, et al. Mechanisms of apoptotic effects induced by resveratrol, dibenzoylmethane and their analogues on human lung carcinoma cells. J Agric Food Chem 2009;57:5235– 43. Li Y, Bäckesjö CM, Haldosén LA, et al. Resveratrol inhibits proliferation and promotes apoptosis of osteosarcoma cells. Eur J Pharmacol 2009;609:13– 8. Rimando AM, Cuendet M, Desmarchelier C, et al. Cancer chemopreventive and antioxidant activities of pterostilbene, a naturally occurring analogue of resveratrol. J Agric Food Chem 2002;50: 3453–7. Ferrer P, Asensi M, Segarra R, et al. Association between pterostilbene and quercetin inhibits metastatic activity of B16 melanoma. Neoplasia 2005;7:37– 47. Tolomeo M, Grimaudo S, Di Cristina A, et al. Pterostilbene and 3=-hydroxypterostilbene are effective apoptosis-inducing agents in MDR and BCR-ABL-expressing leukemia cells. Int J Biochem Cell Biol 2005;37:1709 –26. Pan MH, Chang YH, Badmaev V, et al. Pterostilbene induces apoptosis and cell cycle arrest in human gastric carcinoma cells. J Agric Food Chem 2007;55:7777– 85. Suh N, Paul S, Hao X, et al. Pterostilbene, an active constituent of blueberries, suppresses aberrant crypt foci formation in the azoxymethane-induced colon carcinogenesis model in rats. Clin Cancer Res 2007;13:350 –5. Remsberg CM, Yanez JA, Ohgami Y, et al. Pharmacometrics of pterostilbene: preclinical pharmacokinetics and metabolism, anticancer, antiinflammatory, antioxidant and analgesic activity. Phytother Res 2008;22:169 –79. Schneider JG, Alosi JA, McDonald DE, et al. Effects of pterostilbene on melanoma alone and in synergy with inositol hexaphosphate. Am J Surg 2009;198:679 – 84.
Association of VA Surgeons
Pterostilbene and tamoxifen show an additive effect against breast cancer in vitro Patrick Mannal, M.D.*, Debbie McDonald, B.S., David McFadden, M.D., M.B.A., F.A.C.S. Division of Surgical Oncology, The University of Vermont/Fletcher Allen Health Care, Burlington, VT, USA KEYWORDS: Pterostilbene; Tamoxifen; Breast cancer; Apoptosis
Abstract BACKGROUND: Tamoxifen is widely used for the treatment of breast cancer. Pterostilbene, a bioavailable stilbenoid found in blueberries, has been found to inhibit breast cancer growth in vitro. It was hypothesized that combining pterostilbene with tamoxifen would produce additive effects on estrogen receptor–positive breast cancer cells. METHODS: Two estrogen receptor–positive breast cancer cell lines, MCF7 and ZR-751, were pretreated with graduated doses of pterostilbene for 24 hours, followed by 5 mol/L tamoxifen. MTT proliferation assays and Cell Death Detection ELISAPLUS tests evaluated cell viability and apoptosis. RESULTS: MCF7 cells showed inhibition (10 and 20 mol/L, P ⬍ .001; 30 mol/L, P ⬍ .05) at all time points when combined with tamoxifen. ZR-751 cells showed additive reductions in cell viability (P ⬍ .001). Cell Death Detection ELISAPLUS indicated increased apoptosis (P ⬍ .01). CONCLUSIONS: Pterostilbene shows an additive inhibitory effect on breast cancer cells when combined with tamoxifen, most likely from augmented cancer cell apoptosis. © 2010 Elsevier Inc. All rights reserved.
Breast cancer is the second leading cause of cancer mortality in women,1 with 191,000 new cases this year.2 Major advances have been in the area of adjuvant therapy, specifically selective estrogen receptor (ER) modulators and aromatase inhibitors.3,4 Epidemiologic studies suggest that a diet rich in fruits and vegetables is associated with a reduced risk for a number of common cancers. Phytochemicals are found in plants and have protective or preventive properties. Stilbenes are 1 class of phytochemicals shown to have antioxidant and antiproliferative properties. Resveratrol, a stilbene found in grapes, has been shown to inhibit a variety of primary tumors.5–9 Pterostilbene, an analogue of resveratrol found in blueberries, has been shown to inhibit tumor growth in various models10 –15 and is more bioavailable than * Corresponding author: Tel.: 802-656-3319; fax: 802-656-0680. E-mail address: [email protected] Manuscript received May 18, 2010; revised manuscript July 7, 2010
0002-9610/$ - see front matter © 2010 Elsevier Inc. All rights reserved. doi:10.1016/j.amjsurg.2010.07.022
resveratrol upon oral ingestion. We hypothesized that combining pterostilbene with tamoxifen would produce an additive effect on ER-positive breast cancer in vitro. In addition, we evaluated the aromatase inhibitor anastrozole because of its increasing role as an adjuvant therapy.
Methods Pterostilbene (3,5-dimethoxy-4-hydroxystilbene), 4-hydroxytamoxifen (trans-4-[1-(4-[2-[dimethylamino]ethoxy] phenyl)-2-phenyl-1-butenyl] phenol), anastrozole (2-[3-[1cyano-1-methyl-ethyl]-5-[1H-1,2,4-triazol-1-ylmethyl] phenyl]-2methyl-propanenitrile), and MTT, a tetrazolium dye (thiazolyl blue tetrazolium bromide), were purchased from Sigma-Aldrich (St Louis, MO). Cell Death Detection ELISAPLUS was purchased from Roche (Mannhiem, Germany).
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The American Journal of Surgery, Vol 200, No 5, November 2010
Results Pterostilbene and tamoxifen MTT assay, combined treatments
Figure 1 MCF-7 cells: pterostilbene pretreatment followed by tamoxifen treatment. Combined results were statistically significant (10 mol/L pterostilbene plus 5 mol/L tamoxifen, P ⱕ .001; 20 mol/L pterostilbene plus 5 mol/L tamoxifen, P ⱕ .05; 30 mol/L pterostilbene plus 5 mol/L tamoxifen, P ⫽ NS).
Breast cancer lines (MCF-7, ZR-75-1, and MDA-MB-231) were purchased from American Type Culture Collection (Manassas, VA). For experiments, cells were harvested from culture monolayers at 80% to 90% confluency. ER-positive MCF-7 and ZR-75-1 cells, as well as ERnegative MDA-MB-231 cells, were added to 96-well plates at 104 cells/well and incubated for 24 hours for adherence. After 24 hours, the cells were pretreated with pterostilbene at 10, 20, and 30 mol/L concentrations for 24 hours. After 24 hours of pterostilbene pretreatment, tamoxifen was added to the culture medium for a final concentration of 5 mol/L tamoxifen. Conversely, the MCF-7 and ZR-75-1 cell lines were pretreated with 5 mol/L tamoxifen for 24 hours, followed by the addition of 10, 20, or 30 mol/L pterostilbene. In addition, MCF-7, ZR-75-1, and MDA-MB231 cells were pretreated for 24 hours with 10, 20, and 30 mol/L pterostilbene, followed by 24, 48, and 72 hours of 50 mol/L anastrozole. Similarly, all cell lines were pretreated with 50 mol/L anastrozole for 24 hours, followed by 24, 48, and 72 hours of 10, 20, and 30 mol/L of pterostilbene. Control cells were treated with Dimethyl sulfoxide (DMSO). After 24, 48, and 72 hours, viability was assessed using MTT assay. Cell death pathways were analyzed using the Cell Death Detection ELISAPLUS protocol at 24 hours. Cells were treated with 20 mol/L pterostilbene for 18 hours, 5 mol/L tamoxifen for 18 hours, or the combination of 20 mol/L pterostilbene for 6 hours followed by 5 mol/L tamoxifen for an additional 18 hours. Samples were assessed for necrosis and apoptosis. Statistical comparisons among groups were performed using Student’s t test or analysis of variance, followed by Bonferroni’s posttests for multiple comparisons.
MCF-7 cells had reductions in cell viability with combined treatment of pterostilbene and tamoxifen. With pterostilbene pretreatment, the effect at 24 hours was 39%, 33%, and 31% (P ⬍ .001) of control for 10, 20, and 30 mol/L pterostilbene, respectively. Cells in the 24-hour group, treated with only 5 mol/L tamoxifen, had 58% cell viability. For the 48-hour tamoxifen treatment group, 26%, 24% (P ⬍ .001), and 24% of cells were viable after the combination of 10, 20, and 30 mol/L pterostilbene and 5 mol/L tamoxifen, with cells receiving 5 mol/L tamoxifen having 42% viability. The 72-hour tamoxifen group had 23% (P ⬍ .001), 23% (P ⱕ .05), and 23% viability with 10, 20, and 30 mol/L pterostilbene. Thirty percent of cells treated only with tamoxifen were viable at 72 hours (Fig. 1). With tamoxifen as the pretreatment agent, there were also reductions in cell viability. Cells treated with 10, 20, and 30 mol/L pterostilbene for 24 hours, after tamoxifen, had 50%, 40%, and 37% (P ⬍ .001) viability, respectively. At 48 hours, the same concentrations of pterostilbene produced 29%, 26%, and 25% (P ⬍ .001) viability. The 72hour pterostilbene group had 25%, 24% (P ⬍ .001), and 24% cell viability after tamoxifen pretreatment (Fig. 2). As with the MCF7 cells, ZR-75-1 cells had reductions in number after combined treatment. With pterostilbene pretreatment of 10, 20, and 30 mol/L for 24 hours, followed by 24 hours of 5 mol/L tamoxifen, the cells had 64%, 61%, and 42% (P ⬍ .001) viability, respectively. At 48
Figure 2 MCF-7 cells: tamoxifen pretreatment followed by pterostilbene treatment. Combined results were statistically significant (5 mol/L tamoxifen plus 10 mol/L pterostilbene, P ⱕ .001; 5 mol/L tamoxifen plus 20 mol/L pterostilbene, P ⱕ .001; 5 mol/L tamoxifen plus 30 mol/L pterostilbene, P ⫽ NS).
P. Mannal et al.
Pterostilbene helps tamoxifen fight breast cancer
579
combination treatment resulted in a .4-fold increase in apoptosis (value ⫽ 1.4). Treatment with 20 mol/L pterostilbene resulted in a .4-fold increase in apoptosis (value ⫽ 1.4), while 5 mol/L tamoxifen resulted in a .1-fold increase in apoptosis (value ⫽ 1.1). The combination of agents resulted in a value of 2.6, or a 1.6-fold increase over controls. Regardless of the treatment, the MDA-MB-231 cells did not display any increase in apoptosis, with all 3 categories (20 mol/L pterostilbene, 5 mol/L tamoxifen, and the combination of pterostilbene and tamoxifen) resulting in a value of .7.
Comments Figure 3 ZR-75-1 cells: pterostilbene pretreatment followed by tamoxifen treatment. All combined results were statistically significant (P ⱕ .001).
hours, viability was 51%, 36%, and 22% (P ⬍ .001). At 72 hours, 35%, 14%, and 5% (P ⬍ .001) of cells were viable after tamoxifen (Fig. 3). With 24 hours of tamoxifen pretreatment, the ZR-75-1 cells produced similar results (Fig. 4). The ER-negative cell line, MDA-MB-231, was exposed to pterostilbene pretreatments of 20 and 30 mol/L for 24 hours, followed by 5 mol/L tamoxifen for 24, 48, and 72 hours. Essentially no additive effects were seen, although pterostilbene did inhibit cell growth alone (P ⬍ .01).
Pterostilbene and anastrozole MTT assay, combined treatment
Previous studies have clearly shown the antineoplastic effects of pterostilben.16 We chose to examine the potential additive effects pterostilbene and tamoxifen, a widely used selective ER modulator, in the treatment of ER-positive breast cancer. We also evaluated the combination of anastrozole and pterostilbene. The results of the ER-positive cell line viability assays indicate that the combination of tamoxifen and pterostilbene is additive. At low doses and short time intervals, the 2 agents clearly show a greater effect together than either alone. Our inclusion of the ER-negative MDA-MB-231 cell line helped rule out a bystander effect of the tamoxifen on the target cells. Unfortunately, the mechanism of action wherein pterostilbene acts on target cells is incompletely understood, so an explanation for this effect would be speculative at best. In support of other studies examining the effect pterostilbene on other cancers, the results of our Cell Death Detection ELISAPLUS assays seem to validate an augmented
MCF-7 cells showed no additive reduction in cell viability at any time points or concentrations with combination therapy. In ZR-75-1 cells, 50 mol/L anastrozole pretreatment (24 hours) followed by the increasing doses of pterostilbene did produce significance. At 24 hours, the 10 mol/L pterostilbene treatment group had 80% cell viability (P ⬍ .01), and the 20 mol/L group had 68% viability (P ⬍ .001). After 48 hours of pterostilbene, all 3 groups had reductions to 68%, 55%, and 47% (P ⬍ .001) in response to the 10, 20, and 30 mol/L treatments, respectively. At 72 hours, the 10 mol/L pterostilbene group wase at 61% (P ⬍ .001), and the 20 mol/L group was at 41% (P ⬍ .01).
Pterostilbene and tamoxifen, Cell Death Detection ELISAPLUS Results of the Cell Death Detection ELISAPLUS are provided in fold increases of apoptosis. The MCF-7 cells, using DMSO-treated-only cells as a control, had a .2-fold increase (value ⫽ 1.2) in apoptosis with 20 mol/L pterostilbene treatment. With 5 mol/L tamoxifen treatment, MCF-7 demonstrated no increase in apoptosis (value ⫽ 1), and with
Figure 4 ZR-75-1 cells: tamoxifen pretreatment followed by pterostilbene treatment. All combined results were statistically significant (P ⱕ .001).
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The American Journal of Surgery, Vol 200, No 5, November 2010
apoptotic effect seen in the target cells. There was an obvious increase in apoptosis in the ER-positive cells with no effect on the ER-negative cells. Again, the increase in ERpositive activity was not surprising given the known mechanism of action of tamoxifen. Pterostilbene has been shown to be nontoxic in normal tissues, and our current doses do not exceed physiologic levels of ingestion. Its ability to augment the effects of conventional chemotherapy presents several potential opportunities for its use. First, lower dose conventional therapy may be possible while maintaining its antitumoral effects. This would result in lower costs and an improved side-effect profile. Second, additional antitumoral effects may be seen with conventional doses, thereby extending disease-free survival. In conclusion, the in vitro addition of pterostilbene to tamoxifen resulted in additive antiproliferative effects. Further studies are warranted.
6.
7.
8.
9.
10.
11.
12.
References 1. Altekruse SF, Kosary CL, Krapcho M, et al, eds. SEER cancer statistics review, 1975–2007, National Cancer Institute. Available at: http:// seer.cancer.gov/csr/1975_2007/. 2. US Cancer Statistics Working Group. United States cancer statistics: 1999 –2006 incidence and mortality Web-based report. Available at: http://www.cdc.gov/uscs. 3. Early Breast Cancer Trialists’ Collaborative Group. Effects of chemotherapy and hormonal therapy for early breast cancer on recurrence and 15 year survival on overview of the randomised trials. Lancet 2005;365:1687–717. 4. Osborne CK. Tamoxifen in the treatment of breast cancer. N Engl J Med 1998;339:1609 –18. 5. Lee MH, Choi BY, Kundu JK, et al. Resveratrol suppresses growth of human ovarian cancer cells in culture and in a murine xenograft model:
13.
14.
15.
16.
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