Zeranol may increase the risk of leptin-induced neoplasia in human breast
- Authors:
- Published online on: November 23, 2010 https://doi.org/10.3892/ol.2010.214
- Pages: 101-108
Abstract
Introduction
Breast cancer is a worldwide disease, which over 40,000 women succumb to each year in the US (1). Epidemiological studies suggest that there are numerous risk factors associated with breast cancer, including dietary fat and environmental estrogenic endocrine disruptors.
One of the known risk factors of breast cancer is obesity, which has become a major public health concern (2). The Centers for Disease Control and Prevention reported that about 2/3 of adults in the US are overweight and 1/3 are obese (2–4). The incidence of breast cancer is increased with obesity, and morbidity is increased in obese cancer patients as compared to cancer patients with normal or low weight (2). The relationship between breast cancer and obesity has been studied for more than 40 years (2,5).
Leptin, a transcriptional product of the ob gene, plays a key role in breast cancer development and has been studied since its discovery in 1994 (6). Besides its involvement in appetite regulation and energy balance by sending signals to the hypothalamus (7), leptin has a number of other regulatory functions including ensuring normal mammary gland development, bone development, fetal development, sex maturation, angiogenesis, lactation, hematopoiesis and immune responses (2,3). Additionally, leptin is required for normal mammary gland development in rodents (2,8). Animals and humans with defective leptin or with mutated leptin receptor genes are obese (3,9). In clinical studies, the serum leptin level in prostate cancer patients was found to be higher than that in a healthy control group, and was correlated with prostate-specific antigen (10,11). In a breast cancer research program, 60% (20/35) of the patients expressed leptin, while none out of four cases with normal breast tissue expressed leptin (11,12).
Leptin has a direct mitogenic effect on human breast cancer cells (12); therefore, the inhibition of leptin may contribute to the prevention and treatment of breast cancer (13,14). Leptin expression is up-regulated in obesity (3) and promotes breast cancer cell growth by directly affecting the estrogen receptor (ER) pathway (3). Similar to other growth factors and cytokines, leptin is present in human serum and plays a role in human cancer development (3). Since leptin was found to be associated with breast cancer (3,15), investigators have attempted to determine the relationship and mechanisms of leptin action in breast cancer (16–21). Ishikawa et al found that leptin was overexpressed in breast cancer cells (13) and similarly concluded that high leptin levels in obese breast cancer patients may play a role in the development of antiestrogen resistance (16). Leptin is not expressed in normal breast tissue but exists near malignant breast lesions (12). In addition to its mitogenic effects, leptin promotes T47-D cell line proliferation (14) and a high level of leptin may contribute to the development of a more aggressive malignant phenotype (22). ICI 182,780 is a pure estrogen antagonist approved for the treatment of breast cancer patients who fail to respond to tamoxifen therapy. Treatment of cells with ICI 182,780 resulted in rapid degradation of membrane ER α, which reduced the nuclear expression of the receptor and ER α-dependent transcription, and produced significant growth inhibition. Leptin was able to counteract the cytostatic activity of ICI 182,780 as well as the effect of this compound on the expression of ER α and ER α-dependent transcription (23). The power of leptin to stimulate human MCF-7 cell growth and to counteract the effects of ICI 182,780 strongly suggests that leptin acts as a paracrine/endocrine growth factor towards mammary epithelial cells (16). Chen et al also found that leptin increases ZR-75-1 breast cell growth by up-regulating cyclin D1 and down-regulating P53 (24). Since it stimulates estrogen biosynthesis through the induction of aromatase activity and the modulation of ER α activity, leptin has been characterized as a growth factor for breast cancer (3,25). High levels of leptin in obese breast cancer patients may play a notable role in breast cancer cell proliferation, invasion and metastasis (2).
Estrogen has been regarded as a positive regulator of leptin production (26), and leptin levels in breast cancer patients treated with tamoxifen are significantly higher than those in the control group (27). Zearalenone, a stable natural product that mimics estrogen activity, is a carcinogen and thus hazardous to human health (28). Zeranol (Z), produced from zearalenone, is a non-estrogenic anabolic growth promoter used to stimulate cattle growth in the US beef industry (29). Zearalenone and Z bind to the active site of human ER α and ER β in a similar manner to 17 β-estradiol (30). Since it is a widespread food contaminant, it is difficult to avoid the intake of Z (28). Based on its toxicity information, the FDA approved the use of Z in the beef cattle industry. However, the European Union declined to import beef products with residues of hormonal implantation from the US due to the potential health concerns. It was reported that Z enhanced the proliferation of pre-adipocytes in beef heifers (31). A previous study found that Z did not change the serum leptin level in growing wethers (32). At low concentrations, it increases ER α-positive cell growth, but a high concentration of Z reduces the growth of ER α-positive and -negative cell lines (33). Moreover, previous data showed that Z was able to transform human normal breast epithelial cells and increase human breast cell growth in a dose-dependent manner (29). Additionally, Z down-regulated the estrogen-regulated human breast cancer candidate suppressor gene, protein tyrosine phosphatase γ expression (34).
Gossypol, another natural polyphenolic compound extracted from cottonseed and used as an anticancer chemopreventive agent, inhibits breast cancer cell growth (35,36). It is suggested that gossypol be used as a potential chemopreventive food component. It was also demonstrated that gossypol exhibits anticancer activity against multidrug resistant human breast cancer cells (36,37), with (-)-gossypol having the strongest effect among the three isoforms (data not shown).
This study aimed to investigate the interaction of leptin, Z and gossypol in breast cancer development, as well as the mechanisms involved in the suppression of Z- and leptin-induced proliferation of primary cultured human normal breast epithelial cells using (-)-gossypol as the main chemopreventive agent.
Materials and methods
Animal treatment and blood sampling
Ralgro Magnum® (RM, commercial Z pallet) was purchased from Schering-Plough Corp, Kenilworth, NJ, USA, in the form of cartridges, each containing 72 mg Z. A total of 20 cross-bred Angus beef heifers (~1 year old) were purchased from the Department of Animal Science. The animals were randomly divided into two groups according to initial body weight. Animal treatment was described in our previous publication (31). Z-containing sera (ZS) dropped from the Z-implanted beef at day 0 (ZS-D0, prior to Z implantation) and 30 days post Z implantation (ZS-D30), and non-Z-containing serum (NZS) from non-Z-implanted beef at day 0 (NZS-D0, prior to Z implantation) and 30 days post Z implantation (NZS-D30) were sterilized using a 50 ml conical filter tube, and stored at −20°C.
Tissue culture
Human normal breast tissues were sterilized in 70% ethanol for 30 sec, and then washed three times with fresh Dulbecco’s modified Eagle’s medium and Ham’s F12 medium (DMEM/F12). In vitro organ-cultured human normal breast tissues were treated with leptin at 0, 1.5, 3.0 and 6.0 nM in DMEM/F12 supplemented with 5% dextran-coated charcoal (DCC)-stripped fetal bovine serum (FBS) and cultured in a 10 cm2 cell culture plate in a humidified incubator (5% CO2, 95% air, 37°C) for 96 h. The medium was changed every 48 h.
Primary-cultured human normal breast epithelial cell (HNBEC) isolation
The cultured human normal breast tissues were minced and digested using a digestion buffer consisting of phenol red-free high calcium (DMEM/F12, 1:1) (1.05 mM CaCl2) with 2% bovine serum albumin (BSA) (Invitrogen, Carlsbad, CA, USA) containing 10 ng/ml cholera toxin (Sigma, St. Louis, MO, USA) 6,300 U/ml collagenase (Invitrogen) and 100 U/ml hyaluronidase (Calbiochem, Gibbstown, NJ, USA). The mixture was incubated in a humidified incubator (5% CO2, 95% air, 37°C) overnight, and the solution was transferred to a 50 ml tube and centrifuged at 1,200 rpm for 5 min. The upper, middle and lower layers were separated and centrifuged again. The upper and middle layers, containing pre-adipocytes and stromal cells, respectively, were transferred to another 15-ml tube separately while the lower layer containing epithelial cells remained in the tube. The pellets were washed using DMEM/F12 with antibiotic-antimycotic (100 U/ml penicillin G sodium, 100 μg/ml streptomycin sulfate and 0.25 μg/ml amphotericin B) (Invitrogen) and centrifuged again. The pellets were then washed three times. The final pellet in the tube contained HNBECs and a few stromal cells. The pellet was resuspended in 10 ml low calcium (0.04 mM CaCl2) DMEM/F12 supplemented with 10% of low calcium FBS (Atlanta Biologicals, Norcross, GA, USA) and then transferred into a T75 flask for culturing.
Cell culture
The isolated HNBECs were cultured in a 75 cm2 culture flask in a humidified incubator (5% CO2, 95% air, 37°C) with 10 ml low calcium (0.04 mM CaCl2) DMEM/F12 mixture (Atlanta Biologicals) supplemented with 10% of Chelex-100- (Bio-Rad Laboratories, Richmond, CA, USA) treated FBS (Invitrogen). Low-calcium DMEM/F12 was changed every two days. Only HNBECs survived in this medium; thus, the growth of the stromal cells isolated from the same tissue stopped and the purity of the HNBECs was guaranteed. When the cells reached 85–90% confluence, they were washed with 10 ml of calcium- and magnesium-free phosphate-buffered saline (PBS, pH 7.3), and then trypsinized with 3 ml of 0.25% trypsin-5.3 mM EDTA (Invitrogen) for 10 min at 37°C. Trypsinization was stopped by adding 10 ml of DMEM/F12 with 10% FBS. Following centrifugation, the dissociated cells were re-suspended in low-calcium DMEM/F12 with 10% low-calcium FBS and sub-cultured into 75 cm2 culture flasks at a ratio of 1:5. The experiments were conducted on HNBECs not generated beyond the fourth passage.
Cell proliferation (MTT) assay
A total volume of 100 μl medium containing 4,000 HNBECs/well was seeded in 96-well plates in low-calcium DMEM/F12 and incubated in 37°C for 24 h. The following day, the medium was replaced with 100 μl low-calcium DMEM/F12 supplemented with 0.2% BSA and incubated in 37°C for a further 24 h. Following the treatment, 1.5, 3.0 and 6.0 nM of leptin was administered to the HNBECs isolated from non-leptin treated human normal breast tissues for 0, 6, 12 and 24 h, and 0.1% DMSO was administered to the control group. The proliferation of HNBECs was measured by adding 20 μl of a fresh mixture of 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxy-phenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS) and phenazine methosulfate (PMS) (20:1) solution (Promega, Madison, WI, USA) to the wells. Following incubation at 37°C for 1–5 h, the optical density (OD) values were measured using a kinetic microplate reader (Molecular Devices Cooperation, Menio Park, CA, USA) at a 490 nm wavelength, and the cell growth was compared.
The sera used in the MTT assay were: NZS-D0, NZS-D30, ZS-D0 and ZS-D30. The concentration for NZS and ZS was 0.2, 1.0 and 5.0% in the cultured medium and was administered to HNBECs isolated from the controls, as well as to 1.5 nM leptin-cultured human normal breast tissues for 6 h. The cell proliferation was measured as described above.
To investigate the effect of the combination of leptin with (-)-gossypol in HNBEC growth, 4,000/well HNBECs isolated from non-leptin cultured tissues were seeded in 96-well plates in low-calcium DMEM/F12 and incubated at 37°C for 24 h. The following day, the medium was replaced by 100 μl of low-calcium DMEM/F12 supplemented with 0.2% BSA and incubated at 37°C for a further 24 h. The treatment of 1.5, 3 and 6 nM leptin alone or in combination with 10 μM (-)-gossypol, with 0.1% DMSO as the control was administered, and the proliferation of HNBECs was measured using the methods described above.
Cell treatment for RNA and PCR analysis
Viable HNBECs (105/well) were seeded in 6-well plates in 5 ml low-calcium DMEM/F12 supplemented with 10% low-calcium FBS. After 24 h, the medium was replaced with low-calcium DMEM/F12 supplemented with 10% DCC-stripped low-calcium FBS. The cells were cultured overnight. After 24 h, the medium was changed and 24 h of treatment was administered. The concentration for leptin was 1.5, 3 and 6 nM and that for Z was 5, 10 and 20 nM. The combination of 1.5 nM leptin and 5 nM Z with or without 3 μM (-)-gossypol was also administered, with 0.1% DMSO being administered to the control group.
RNA isolation, cDNA synthesis and reverse transcription polymerase chain reaction (RT-PCR)
Following the treatment of HNBECs for 24 h, the cultured medium was collected for leptin measurement. Total RNA was isolated in 1 ml TRIzol reagent (Invitrogen) according to the manufacturer’s instructions (37). The RT-PCR conditions were optimized for each primer and performed using a thermocycler Gene Amp PCR (Eppendorf®, Westbury, NY, USA). A volume of 2 μl of the newly-synthesized cDNA was used as a template for RT-PCR. The PCR conditions were optimized for the MgCl2 concentration, annealing temperature and cycle number for the amplification of each of the PCR products (cyclin D1, 36B4). Under optimal conditions, 1 unit of platinum Taq DNA polymerase (Invitrogen) was added for a total volume of 25 μl.
The primers for cyclin D1 were: upper, 5′-GCT CCT GTG CTG CGA AGT GG-3′ and lower, 5′-TGG AGG CGT CGG TGT AGA TG-3′ (product size 372 bp). The PCR conditions were: denaturing at 95°C for 5 min, followed by 27 cycles at 94°C for 45 sec, 54°C for 45 sec, 72°C for 60 sec and extension at 72°C for 10 min. The primers for 36B4 were: upper, 5′-AAA CTG CTG CCT CAT ATC CG-3′ and lower, 5′-TTT CAG CAA GTG GGA AGG TG-3′ (product size 563 bp). The PCR conditions were: denaturing at 95°C for 5 min, followed by 24 cycles at 95°C for 60 sec, 63°C for 60 sec, 72°C for 60 sec and extension at 72°C for 10 min. Pure H2O was used as a negative control to detect genomic DNA contamination and 36B4 as the internal control whose RNA is unmodified by treatment.
The final RT-PCR products (10 μl) mixed with 1 μl 10X loading buffer were separated on 1.5% agarose gel and visualized by staining with ethidium bromide. Electronic images were captured by a Fujifilm LAS-3000 image system (Fuji Film Medical Systems USA, Inc. Stanford, CT, USA). The densities of specific bands were quantified by ImageQuant software (Molecular Dynamics, Sunnyvale, CA, USA). The results were presented as the ratio of cyclin D1 to 36B4.
Western blotting assay
HNBECs isolated from non-leptin cultured tissue were plated in a 10-cm2 culture dish with a density of 1×106 viable cells/well with a 10 ml low-calcium DMEM/F12 supplemented with 10% low-calcium FBS and cultured overnight. The medium was then replaced with low-calcium DMEM/F12 supplemented with 10% DCC-treated low-calcium FBS and cultured for a further 24 h. The primary cultured HNBECs were then treated with 1.5 and 3 nM leptin, 5, 10 and 20 nM Z or 0.1% DMSO as a vehicle control. Following 24 h of treatment, culture media were collected for the leptin measurement and proteins were isolated from the control and treatment groups using M-PER® mammalian protein extraction reagent (Pierce, Rockford, IL, USA) according to the manufacturer’s instructions. Culture media were collected for the leptin measurement, and 400 μl of extraction reagent was added to each dish. Each dish was then placed on an orbital agitator to be digested for 5 min. The digested products were collected and transferred to a 1.5-ml centrifuge tube. The mixture was centrifuged at 10,000 × g for 5 min, and the supernatant was then transferred to a new 1.5-ml centrifuge tube. The protein concentrations were measured using a Micro BCATM protein assay reagent kit (Pierce) according to the manufacturer’s instructions. Proteins (50 μg) from each treatment group were separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis and transferred to a polyvinylidine fluoride membrane (Bio-Rad Laboratory, Hercules, CA, USA). The membrane was initially blotted in PBS-Tween 20 (PBST) containing 10% fat-free dry milk for 1 h and then incubated with primary antibody (cyclin D1 1:1,000 dilution, Cell Signaling Technology® Danvers, MA, USA; β actin, 1:2,000 dilution, Santa Cruz Biotechnology, Inc. Santa Cruz, CA, USA) for 1 h. The membrane was rinsed in PBST three times, each time for 5 min. The membrane was then incubated with the second antibody for 1 h. After the membrane had been washed three times in PBST, it was detected using the Fuji Imaging System (Fuji Film Medical Systems USA, Inc.). The images were captured by FujiFilm LAS-300 image system (Fuji Film). The protein ratios of cyclin D1 to β actin were calculated by measuring the density of the specific band using Multi Gauge (V3.0) software.
Leptin measurement
The culture media were collected prior to protein extraction as previously described. The human leptin immunoassay kit for leptin measurement was purchased from R&D Systems (Minneapolis, MN, USA). The particles in the cell culture supernates were removed by centrifugation at 1,200 rpm for 5 min and the assay was performed immediately according to the manufacturer’s instructions. The assay was stopped by the addition of 50 μl of stop solution to each well. The OD values were then measured within 30 min using a kinetic microplate reader (Molecular Devices Cooperation, Menio Park, CA, USA) at a wavelength of 450 nm and the leptin concentration was compared.
Statistical analysis
The results for the cell proliferation assay are presented as mean ± standard deviation (SD) of 4 replicate culture wells. The analysis was performed using Minitab 15 (Minitab Inc., PA, USA). The statistical difference was determined using two sample t-test analyses for independent samples. P<0.05 was considered to be statistically significant.
Results
Leptin increased HNBEC proliferation
As shown in Fig. 1A, leptin increased the growth of HNBECs. When compared to their respective control groups, it was found that 3 nM leptin with 24 h of treatment or 6 nM leptin with 12 h of treatment significantly increases HNBEC growth. Fig. 1B shows that (-)-gossypol inhibits HNBEC growth induced by 3 and 6 nM leptin. A significant difference between leptin and the combination of leptin with (-)-gossypol is noted.
Z-containing serum increases primary cultured HNBEC growth
Fig. 2A shows that NZS-D0 and NZS-D30 have no effect on HNBECs isolated from non-leptin cultured tissue at any dose, but stimulated HNBEC growth isolated from leptin cultured tissue at the same dose (Fig. 2B).
Fig. 2C shows that ZS-D0 and ZS-D30 at concentrations of 0.2, 1 and 5% increased HNBECs isolated from non-leptin cultured tissue as compared to the control group, respectively. Fig. 2D shows that ZS-D0 and ZS-D30 at concentrations of 0.2, 1 and 5% increased HNBECs isolated from 1.5 nM of leptin cultured tissue with a significant difference between ZS-D0 and ZS-D30 at all doses.
The effect of leptin, zeranol and (-)-gossypol in the cyclin D1 expression in breast cells
HNBECs were treated with 1.5, 3 and 6 nM leptin, 5, 10 and 20 nM Z, a combination of 5 nM Z and 1.5 nM leptin with or without 3 μM (-)-gossypol for 24 h. Significant differences in cyclin D1 expression were found between the control group and the group treated with 3 and 6 nM leptin and 10 and 20 nM Z (p<0.05). In addition, the combination of 1.5 nM leptin with 5 nM Z significantly increased HNBEC growth as compared to the control group. However, the effective combination was inhibited by adding 3 μM (-)-gossypol (Fig. 3A). Consistent with the mRNA expression, cyclin D1 protein expression also increased with the 24 h treatment of Z in HNBECs (Fig. 3B).
Zeranol increased HNBEC leptin secretion
As shown in Fig. 4, 20 nM Z significantly increased leptin secretion in HNBECs, as compared to the control group (p<0.05).
Discussion
Cyclin D1 is a cell cycle regulator that plays an important role in cell growth. It is the product of the CCND1 gene which is located on chromosome 11q13 (39). The cyclin-dependent kinases (CDKs) cannot modulate cell growth without the cyclin subunit. By binding to cyclin D, cyclin D-CDK 4/6 comprises the mechanism of the cell cycle and affects the G1 phase in cell growth. The cyclin D1 level is modulated by changing growth factors in the medium used to culture the cells. Cyclin D1 was found to be overexpressed in over 50% of breast cancer patients and is known as one of the most overexpressed proteins in breast cancer (39). Leptin stimulates breast cancer cell growth by up-regulating the cyclin D1 expression. Moreover, Garofalo et al noted that leptin regulates estrogen synthesis and ER α activity (3). Besides regulating the cell cycle, it was noted that cyclin D1 correlates with ER (39). Cyclin D1 binds to ER and stimulates its transcriptional activities. The cyclin D1 and ER complex may play a role in stimulating tumor cell proliferation. It is crucial to elucidate whether leptin and Z affect the cyclin D1 expression in primary cultured normal breast epithelial cells.
Results of this study show that 6 nM leptin or 30 nM Z alone had no effect on the cyclin D1 expression. However, their combination significantly increased the cyclin D1 expression as compared to the control group. In our cell proliferation assay, cells isolated from leptin exposure tissues increased their sensitivity to Z. This increase is partially explained by the fact that the combination resulted in a high expression of cyclin D1. A previous study noted that the serum level of leptin in breast cancer patients is higher than that in controls (27). It is likely that if obese healthy women or breast cancer patients have higher leptin in their serum, the sensitivity of normal or cancerous breast cells is increased to the presence of Z in beef. Consequently, these results suggest a possible correlation between obesity and breast neoplasia and indicate a potential risk for breast neoplasia in obese individuals, particularly in those consuming beef from animals implanted with Z.
Of noteis that leptin secretion was increased by the treatment of Z in HNBECs; thus, Z amplified the leptin activity. It was reported that leptin affects transformed breast cancer cells to induce an alteration to a more aggressive phenotype, and leptin potentially serves as a tumor marker. This result shows that Z is potentially more harmful to obese individuals than those with normal weight by increasing the risk of breast neoplasia (33) since the consumption of Z-containing products by the obese individuals increases their chances of developing breast neoplasia. However, (-)-gossypol has been found to reverse the effect of the combination of leptin with Z on cell growth and may therefore be used in the treatment of breast cancer patients.
The effect of ZS in human normal breast epithelial cell growth was also evaluated. We found that 2.5% of ZS harvested from 60-day post 72 mg Z pellet-implanted beef heifers transformed the human normal breast epithelial cell line, MCF-10A, to breast neoplasia cells in a 21-day culture (unpublished data). Our current data showed that there was no observable proliferative stimulation from exposure to NZS for 6 h in HNBECs isolated from the non-leptin cultured tissues (Fig. 2A). However, the proliferation of HNBECs isolated from 1.5 nM leptin cultured tissues was significantly increased by treatment of ZS at all doses for 6 h (Fig. 2D). As shown in Figs. 2C and D, ZS increases HNBECs isolated from leptin cultured tissues more than that isolated from non-leptin cultured tissues. A comparison of Fig. 2A to Fig. 2B and Fig. 2C to Fig. 2D showed that HNBECs isolated from 1.5 nM of leptin cultured tissues developed more rapidly than those isolated from non-leptin cultured tissues with the same treatment of ZS or NZS. This result shows that leptin stimulates HNBEC growth.
On the other hand, a comparison of Fig. 2A to Fig. 2C and Fig. 2B to Fig. 2D showed that ZS increases HNBECs isolated from leptin cultured tissues more than those isolated from non-leptin cultured tissues. The stimulatory effect of ZS is greater than that of NZS in HNBECs isolated from with or without leptin cultured tissues. A significant difference between the ZS-D0 and ZS-D30 at concentrations of 0.2, 1 and 5% was only found in the HNBECs isolated from 1.5 nM of leptin cultured tissues. The only difference between NZS and ZS was the implantation of Z pellets and the presence of metabolites in the blood. This result suggests that certain as yet undefined growth factors responsible for stimulatory action in HNBEC proliferation are secreted by the Z-implanted heifers in the blood. Therefore, we attribute the stimulatory effect of ZS on HNBECs to the implantation of Z.
It appears that leptin stimulates HNBEC growth, while ZS-D30 improves leptin-induced growth. Considering the leptin level is higher in obese women than in normal or lower weight women, this result suggests that obese women are more sensitive to Z. Additionally, it was shown that obese women may have a higher risk of breast neoplasia due to the consumption of beef products containing Z.
In conclusion, we found that the mitogenic activity of Z in human normal breast epithelial cells is enhanced by leptin and inhibited by gossypol. Z appears to increase HNBEC growth by increasing the cyclin D1 expression. Leptin improves HNBEC sensitivity to Z and Z strengthens the effect of leptin by increasing leptin secretion in HNBECs. Leptin and Z up-regulate the cyclin D1 expression in HNBECs. However, (-)-gossypol counteracts the growth of breast cancer cells induced by leptin alone or in combination with Z by down-regulating the cyclin D1 mRNA expression. Further mechanisms are currently being investigated and research on the Z metabolites that stimulate HNBEC growth is ongoing.
Acknowledgements
This study was supported by NIH grant R01 ES 015212.
References
Jemal A, Siegel R, Ward E, Hao Y, Xu J and Thun MJ: Cancer statistics. CA Cancer J Clin. 59:225–249. 2009. | |
Calle EE and Thun MJ: Obesity and cancer. Oncogene. 23:6365–6378. 2004. View Article : Google Scholar | |
Garofalo C and Surmacz E: Leptin and cancer. J Cell Physiol. 207:12–22. 2006. View Article : Google Scholar | |
Lorincz AM and Sukumar S: Molecular links between obesity and breast cancer. Endocr Relat Cancer. 13:279–292. 2006. View Article : Google Scholar : PubMed/NCBI | |
De Waard F, Baanders-Vanhalewijn EA and Huizinga J: The bimodal age distribution of patients with mammary carcinoma; evidence for the existence of 2 types of human breast cancer. Cancer. 17:141–151. 1964.PubMed/NCBI | |
Zhang Y, Proenca R, Maffei M, Barone M, Leopold L and Friedman JM: Positional cloning of the mouse obese gene and its human homologue. Nature. 372:425–432. 1994. View Article : Google Scholar : PubMed/NCBI | |
Rosen ED and Spiegelman BM: Adipocytes as regulators of energy balance and glucose homeostasis. Nature. 444:847–853. 2006. View Article : Google Scholar : PubMed/NCBI | |
Kiess W, Petzold S, Topfer M, et al: Adipocytes and adipose tissue. Best Pract Res Clin Endocrinol Metab. 22:135–153. 2008. View Article : Google Scholar | |
Clement K, Vaisse C, Lahlou N, et al: A mutation in the human leptin receptor gene causes obesity and pituitary dysfunction. Nature. 392:398–401. 1998. View Article : Google Scholar : PubMed/NCBI | |
Buschemeyer WC III and Freedland SJ: Obesity and prostate cancer: epidemiology and clinical implications. Eur Urol. 52:331–343. 2007. View Article : Google Scholar : PubMed/NCBI | |
Caldefie-Chezet F, Damez M, de Latour M, et al: Leptin: a proliferative factor for breast cancer? Study on human ductal carcinoma. Biochem Biophys Res Commun. 334:737–741. 2005. View Article : Google Scholar : PubMed/NCBI | |
Rose DP, Komninou D and Stephenson GD: Obesity, adipocytokines, and insulin resistance in breast cancer. Obes Rev. 5:153–165. 2004. View Article : Google Scholar : PubMed/NCBI | |
Ishikawa M, Kitayama J and Nagawa H: Enhanced expression of leptin and leptin receptor (OB-R) in human breast cancer. Clin Cancer Res. 10:4325–4331. 2004. View Article : Google Scholar : PubMed/NCBI | |
Laud K, Gourdou I, Pessemesse L, Peyrat JP and Djiane J: Identification of leptin receptors in human breast cancer: functional activity in the T47-D breast cancer cell line. Mol Cell Endocrinol. 188:219–226. 2002. View Article : Google Scholar : PubMed/NCBI | |
Calle EE, Rodriguez C, Walker-Thurmond K and Thun MJ: Overweight, obesity, and mortality from cancer in a prospectively studied cohort of U.S. adults. N Engl J Med. 348:1625–1638. 2003. View Article : Google Scholar : PubMed/NCBI | |
Garofalo C, Sisci D and Surmacz E: Leptin interferes with the effects of the antiestrogen ICI 182,780 in MCF-7 breast cancer cells. Clin Cancer Res. 10:6466–6475. 2004. View Article : Google Scholar : PubMed/NCBI | |
Catalano S, Mauro L, Marsico S, et al: Leptin induces, via ERK1/ERK2 signal, functional activation of estrogen receptor alpha in MCF-7 cells. J Biol Chem. 279:19908–19915. 2004. View Article : Google Scholar : PubMed/NCBI | |
Frankenberry KA, Skinner H, Somasundar P, McFadden DW and Vona-Davis LC: Leptin receptor expression and cell signaling in breast cancer. Int J Oncol. 28:985–993. 2006.PubMed/NCBI | |
Saxena NK, Vertino PM, Anania FA and Sharma D: Leptin-induced growth stimulation of breast cancer cells involves recruitment of histone acetyltransferases and mediator complex to CYCLIN D1 promoter via activation of Stat3. J Biol Chem. 282:13316–13325. 2007. View Article : Google Scholar | |
Catalano S, Marsico S, Giordano C, Mauro L, Rizza P, Panno ML and Ando S: Leptin enhances, via AP-1, expression of aromatase in the MCF-7 cell line. J Biol Chem. 278:28668–28676. 2003. View Article : Google Scholar : PubMed/NCBI | |
Yin N, Wang D, Zhang H, et al: Molecular mechanisms involved in the growth stimulation of breast cancer cells by leptin. Cancer Res. 64:5870–5875. 2004. View Article : Google Scholar : PubMed/NCBI | |
Goodwin PJ, Ennis M, Fantus IG, Pritchard KI, Trudeau ME, Koo J and Hood N: Is leptin a mediator of adverse prognostic effects of obesity in breast cancer? J Clin Oncol. 23:6037–6042. 2005. View Article : Google Scholar : PubMed/NCBI | |
Cirillo D, Rachiglio AM, la Montagna R, Giordano A and Normanno N: Leptin signaling in breast cancer: an overview. J Cell Biochem. 105:956–964. 2008. View Article : Google Scholar : PubMed/NCBI | |
Chen C, Chang YC, Liu CL, Chang KJ and Guo IC: Leptin-induced growth of human ZR-75-1 breast cancer cells is associated with up-regulation of cyclin D1 and c-Myc and down-regulation of tumor suppressor p53 and p21WAF1/CIP1. Breast Cancer Res Treat. 98:121–132. 2006. View Article : Google Scholar : PubMed/NCBI | |
Garofalo C, Koda M, Cascio S, et al: Increased expression of leptin and the leptin receptor as a marker of breast cancer progression: possible role of obesity-related stimuli. Clin Cancer Res. 12:1447–1453. 2006. View Article : Google Scholar | |
Thorn SR, Meyer MJ, Van Amburgh ME and Boisclair YR: Effect of estrogen on leptin and expression of leptin receptor transcripts in prepubertal dairy heifers. J Dairy Sci. 90:3742–3750. 2007. View Article : Google Scholar : PubMed/NCBI | |
Ozet A, Arpaci F, Yilmaz MI, et al: Effects of tamoxifen on the serum leptin level in patients with breast cancer. Jpn J Clin Oncol. 31:424–427. 2001. View Article : Google Scholar : PubMed/NCBI | |
Coe JE, Ishak KG, Ward JM and Ross MJ: Tamoxifen prevents induction of hepatic neoplasia by zeranol, an estrogenic food contaminant. Proc Natl Acad Sci USA. 89:1085–1089. 1992. View Article : Google Scholar : PubMed/NCBI | |
Liu S and Lin YC: Transformation of MCF-10A human breast epithelial cells by zeranol and estradiol-17beta. Breast J. 10:514–521. 2004. View Article : Google Scholar : PubMed/NCBI | |
Takemura H, Shim JY, Sayama K, Tsubura A, Zhu BT and Shimoi K: Characterization of the estrogenic activities of zearalenone and zeranol in vivo and in vitro. J Steroid Biochem Mol Biol. 103:170–177. 2007. View Article : Google Scholar : PubMed/NCBI | |
Ye W, Xu P, Threlfall WR, et al: Zeranol enhances the proliferation of pre-adipocytes in beef heifers. Anticancer Res. 29:5045–5052. 2009.PubMed/NCBI | |
Narro LA, Thomas MG, Silver GA, Rozeboom KJ and Keisler DH: Body composition, leptin, and the leptin receptor and their relationship to the growth hormone (GH) axis in growing wethers treated with zeranol. Domest Anim Endocrinol. 24:243–255. 2003. View Article : Google Scholar : PubMed/NCBI | |
Yuri T, Tsukamoto R, Miki K, Uehara N, Matsuoka Y and Tsubura A: Biphasic effects of zeranol on the growth of estrogen receptor-positive human breast carcinoma cells. Oncol Rep. 16:1307–1312. 2006.PubMed/NCBI | |
Liu S, Sugimoto Y, Sorio C, Tecchio C and Lin YC: Function analysis of estrogenically regulated protein tyrosine phosphatase gamma (PTPgamma) in human breast cancer cell line MCF-7. Oncogene. 23:1256–1262. 2004. View Article : Google Scholar : PubMed/NCBI | |
Gilbert NE, O’Reilly JE, Chang CJ, Lin YC and Brueggemeier RW: Antiproliferative activity of gossypol and gossypolone on human breast cancer cells. Life Sci. 57:61–67. 1995. View Article : Google Scholar : PubMed/NCBI | |
Ye W, Chang HL, Wang LS, et al: Modulation of multidrug resistance gene expression in human breast cancer cells by (-)-gossypol-enriched cottonseed oil. Anticancer Res. 27:107–116. 2007.PubMed/NCBI | |
Xu P, Ye W, Jen R, Lin SH, Kuo CT and Lin YC: Mitogenic activity of zeranol in human breast cancer cells is enhanced by leptin and suppressed by gossypol. Anticancer Res. 29:4621–4628. 2009.PubMed/NCBI | |
Lorincz AM and Sukumar S: Molecular links between obesity and breast cancer. Endocr Relat Cancer. 13:279–292. 2006. View Article : Google Scholar : PubMed/NCBI | |
Roy PG and Thompson AM: Cyclin D1 and breast cancer. Breast. 15:718–727. 2006. View Article : Google Scholar : PubMed/NCBI |