This article was downloaded by: [Memorial University of Newfoundland] On: 26 January 2015, At: 00:55 Publisher: Routledge Informa Ltd Registered in England and Wales Registered Number: 1072954 Registered office: Mortimer House, 37-41 Mortimer Street, London W1T 3JH, UK

Nutrition and Cancer Publication details, including instructions for authors and subscription information: http://www.tandfonline.com/loi/hnuc20

Leptin Induces a Proliferative Response in Breast Cancer Cells but Not in Normal Breast Cells a

ab

a

a

Virginie Dubois , Thierry Jardé , Laetitia Delort , Hermine Billard , Dominique Bernardc

d

d

Gallon , Emmanuelle Berger , Alain Geloen , Marie-Paule Vasson Chezet

ae

& Florence Caldefie-

ae

a

Clermont-Université, Université d’Auvergne, Unité de Nutrition Humaine, BP10448, F-63000 Clermont-Ferrand, France and INRA, UMR 1019, UNH, ECREIN, CRNH Auvergne, Clermont-Ferrand, France

Click for updates

b

Department of Anatomy and Developmental Biology, Monash University, Clayton, Australia

c

Centre Jean-Perrin, EA 4677, Clermont-Ferrand, France

d

INSERM U.1060/INRA U. 1235/INSA, Lyon, France

e

CLARA, Région Lyon Auvergne Rhône-Alpes, France Published online: 16 Apr 2014.

To cite this article: Virginie Dubois, Thierry Jardé, Laetitia Delort, Hermine Billard, Dominique Bernard-Gallon, Emmanuelle Berger, Alain Geloen, Marie-Paule Vasson & Florence Caldefie-Chezet (2014) Leptin Induces a Proliferative Response in Breast Cancer Cells but Not in Normal Breast Cells, Nutrition and Cancer, 66:4, 645-655, DOI: 10.1080/01635581.2014.894104 To link to this article: http://dx.doi.org/10.1080/01635581.2014.894104

PLEASE SCROLL DOWN FOR ARTICLE Taylor & Francis makes every effort to ensure the accuracy of all the information (the “Content”) contained in the publications on our platform. However, Taylor & Francis, our agents, and our licensors make no representations or warranties whatsoever as to the accuracy, completeness, or suitability for any purpose of the Content. Any opinions and views expressed in this publication are the opinions and views of the authors, and are not the views of or endorsed by Taylor & Francis. The accuracy of the Content should not be relied upon and should be independently verified with primary sources of information. Taylor and Francis shall not be liable for any losses, actions, claims, proceedings, demands, costs, expenses, damages, and other liabilities whatsoever or howsoever caused arising directly or indirectly in connection with, in relation to or arising out of the use of the Content. This article may be used for research, teaching, and private study purposes. Any substantial or systematic reproduction, redistribution, reselling, loan, sub-licensing, systematic supply, or distribution in any form to anyone is expressly forbidden. Terms & Conditions of access and use can be found at http:// www.tandfonline.com/page/terms-and-conditions

Nutrition and Cancer, 66(4), 645–655 C 2014, Taylor & Francis Group, LLC Copyright  ISSN: 0163-5581 print / 1532-7914 online DOI: 10.1080/01635581.2014.894104

Leptin Induces a Proliferative Response in Breast Cancer Cells but Not in Normal Breast Cells Virginie Dubois Clermont-Universit´e, Universit´e d’Auvergne, Unit´e de Nutrition Humaine, BP10448, F-63000 Clermont-Ferrand, France and INRA, UMR 1019, UNH, ECREIN, CRNH Auvergne, Clermont-Ferrand, France

Downloaded by [Memorial University of Newfoundland] at 00:55 26 January 2015

Thierry Jard´e Clermont-Universit´e, Universit´e d’Auvergne, Unit´e de Nutrition Humaine, BP10448, F-63000 Clermont-Ferrand, France, INRA, UMR 1019, UNH, ECREIN, CRNH Auvergne, Clermont-Ferrand, France, and Department of Anatomy and Developmental Biology, Monash University, Clayton, Australia

Laetitia Delort and Hermine Billard Clermont-Universit´e, Universit´e d’Auvergne, Unit´e de Nutrition Humaine, BP10448, F-63000 Clermont-Ferrand, France and INRA, UMR 1019, UNH, ECREIN, CRNH Auvergne, Clermont-Ferrand, France

Dominique Bernard-Gallon Centre Jean-Perrin, EA 4677, Clermont-Ferrand, France

Emmanuelle Berger and Alain Geloen INSERM U.1060/INRA U. 1235/INSA, Lyon, France

Marie-Paule Vasson Clermont-Universit´e, Universit´e d’Auvergne, Unit´e de Nutrition Humaine, BP10448, F-63000 Clermont-Ferrand, France and INRA, UMR 1019, UNH, ECREIN, CRNH Auvergne, Clermont-Ferrand, France, Centre Jean-Perrin, Clermont-Ferrand, France, and CLARA, R´egion Lyon Auvergne Rhˆone-Alpes, France

Florence Caldefie-Chezet Clermont-Universit´e, Universit´e d’Auvergne, Unit´e de Nutrition Humaine, BP10448, F-63000 Clermont-Ferrand, France, INRA, UMR 1019, UNH, ECREIN, CRNH Auvergne, Clermont-Ferrand, France, and CLARA, R´egion Lyon Auvergne Rhˆone-Alpes, France

Obesity is a risk factor for breast cancer in postmenopausal women. Leptin, a hormone excessively produced during obesity, is suggested to be involved in breast cancer. The aim of the study was to investigate procarcinogenic potential of leptin by evaluating influence of leptin on cell proliferation, cell cycle, apoptosis, and

Submitted 15 November 2012; accepted in final form 31 January 2014. Address correspondence to Florence Caldefie-Ch´ezet, Facult´e de Pharmacie, Laboratoire, SVFp, 28 place Henri-Dunant, B.P. 38 63000, Clermont-Ferrand, France. Phone: +33473177971. Fax: +33473178438. E-mail: [email protected]

signaling on numerous breast cells lines, including 184B5 normal cells, MCF10A fibrocystic cells and MCF-7, MDA-MB-231, and T47D cancer cells. Expressions of leptin and Ob-R were analyzed using qRT-PCR and immunohistochemistry, proliferation using fluorimetric resazurin reduction test and xCELLigence system, apoptosis and cell cycle by flow cytometry, and effect of leptin on different signalling pathways using qRT-PCR and Western blot. Cells were exposed to increasing concentrations of leptin. All cell lines expressed mRNA and protein of leptin and Ob-R. Leptin stimulated proliferation of all cell lines except for 184B5 and MDA-MB-231 cells. Leptin inhibited apoptosis but didn’t alter proportion of cells within cell cycle in MCF7 cells. Leptin induced overexpression of leptin, Ob-R, estrogen receptor, and aromatase mRNA in MCF-7 and T47D cells. Autoregulation induced by leptin, relationship with estrogen pathway, and proliferative and

645

646

V. DUBOIS ET AL.

Downloaded by [Memorial University of Newfoundland] at 00:55 26 January 2015

antiapoptic activity in breast cancer cells may explain that obesityassociated hyperleptinemia may be a breast cancer risk factor.

INTRODUCTION Obesity, considered as a worldwide epidemic phenomenon, is related to various metabolic disorders and is notably involved in cancer development. It is well recognized that obesity is a risk factor for breast cancer among postmenopausal women (1–3). In addition, high body mass index is related to higher rates of breast cancer recurrence and mortality. In this way, overweight or obese women with breast carcinoma were 2.5 times as likely to die of their disease within 5 yr of diagnosis compared with lean women (4). However, the relationship between obesity and breast cancer is not fully understood. High serum estrogen levels observed in obese women, derived from high androgen aromatization by expanded adipose tissue, are considered as a potential factor (5,6). However, this hypothesis does not fully explain the connection between obesity and breast cancer, suggesting that other factors are involved. The adipose tissue, initially considered as a fat storing tissue, is an endocrine organ secreting numerous hormones, called adipokines, such as leptin. Leptin, a 16-kDa peptide hormone, is mainly produced by adipocytes and its physiological serum concentration is 10 ng/ml. Hyperleptinemia is a common feature of obese women, leptin levels increasing as body weight and fat mass increase (7,8). Liuzzi et al. reported that serum leptin concentrations ranged from 15 to 170 ng/ml in 284 obese women (9). Similarly, Cl´ement et al. noted that leptin levels in genetically leptin receptor-deficient obese women reached 670 ng/ml (10). Leptin exerts its activity through binding to the leptin receptor (Ob-R), which show structural similarity to the class I cytokine receptor family. Alternative mRNA splicing gives rise to different isoforms of Ob-R (Ob-Rt representing all the spliced variants of Ob-R), which have a similar extracellular domain but a variable intracellular structure. Ob-Rl, which contains the fulllength intracellular domain, has complete signalling capacities and appears to be particularly important for weight regulating effect by leptin. Recently, it has been suggested that leptin pathway may be involved in breast cancer. Most epidemiological studies show that leptin in women with breast tumor is higher than that of healthy women (3,11 –15). Taking into account BMI, it was found that blood levels of leptin was higher in patients with cancer in obesity than normal weight (16). A positive correlation was found between leptin and tumor size (17) and hyperleptinemia appears to be a negative prognostic factor associated with the presence of metastases and a low rate of survival (3). Futhermore, we previously observed, using an immunohistochemical approach, that leptin and Ob-Rt are strongly coexpressed in breast cancer tissue and that Ob-Rt expression is positively correlated with tumor diameter (18,19). In the same way, Miyoshi et al. noted

that leptin and Ob-Rl mRNA are expressed in breast cancer and that high expression of Ob-R mRNA in breast cancer tissue is associated with poor prognosis for patients with high serum leptin levels (20). Mice Lep-/- (ob/ob), deficient in leptin, do not develop mammary tumors induced while the mouse Lep+/+ and Lep+/− develop respectively breast cancer in 50% and 67% of cases (21). Similarly, mice lacking the receptor Ob-R-/- (db/db) did not induced breast cancer while the mouse Ob-R+/+ and ObR+/− have tumors in, respectively, 69% and 82% of cases (22). In addition, in the ob/ob mice implanted with cancer cells from MMTV-Wnt1 mice spontaneously developing tumors, tumor size is reduced compared to control mice (23). In contrast, in the db/db mice having undergone the same location, the tumor volume is greater than that of control mice. The presence of leptin and its receptor seems to play a role in mammary carcinogenesis (21,22). These proliferative effects of leptin are found in a nude mouse model implanted with MCF7 cells, in which the injection of leptin caused an increase in tumor volume and overexpression of STATs and MAPK signaling pathways (24). The injection of an agonist of the leptin receptor in mice implanted with human breast cancer cells slows tumor growth (25, 26). Numerous in vitro studies also explored the relationship between leptin and breast cancer and have shown that leptin was a proliferative factor in breast cancer cell lines (27–31). However, to the best of our knowledge, only few study explored the effect of leptin on estrogen-independent MDA-MB-231 breast cancer cells and none on MCF10A fibrocystic breast cell line or 184B5 normal breast cell line (31–34). The activity of leptin on estrogen receptor (ER) negative breast cancer cells is still controversial (31,32). In addition, few studies explored the effect of leptin on its own pathway. Thus, Chen et al. noted that leptin treatment induced leptin and Ob-Rl upregulation (17). Estrogen pathway is well known to be of clinical relevance in breast cancer and, interestingly, the relationship between leptin and estrogen pathways seems of particular interest. We have previously observed that Ob-R expression is positively correlated with estrogen receptor (ER) expression in breast cancer tissue (18). Using an in vitro approach, Catalano et al. described that leptin enhances expression of aromatase, an enzyme catalyzing estrogen biosynthesis, and induces activation of estrogen receptor α (ERα) in MCF-7 cells (35,36). Consequently, in the present study, we explored the expression of leptin and Ob-R in estrogen-dependent breast cancer cell lines MCF7 and T47D, in estrogen-independent breast cancer cell line MDA-MB-231, in fibrocystic breast cell line MCF10A and in normal breast cell line 184B5. The influence of leptin on proliferation, apoptosis, and cell cycle was assessed using increasing concentrations of leptin corresponding to human normal or obesity-associated circulating levels. We also investigated the effect of leptin on estrogen pathway and on its own pathway.

LEPTIN PROLIFERATIVE RESPONSE IN BREAST CANCER CELLS

Downloaded by [Memorial University of Newfoundland] at 00:55 26 January 2015

MATERIALS AND METHODS Cell Culture Human normal breast cells 184B5, human fibrocystic breast cells MCF10A, and human breast cancer cells MCF-7, MDAMB-231, and T47D were obtained from the American Type Culture Collection (ATCC). The 184B5 cells were cultured in MEBM supplemented medium. The MCF10A cells were maintained in DMEM HAM’s F12 medium supplemented with 10% heat-inactivated horse serum (HS), EGF (0.02 μg/ml), cholera toxin (0.1 μg/ml), hydrocortisone (0.5 μg/ml), insulin (0.25 UI/ml), and L-glutamine (2 mM). The MCF-7 and T47D cells were maintained in RPMI 1640 supplemented with 10% heat-inactivated FCS, L-glutamine (2 mM), and gentamycin (50 μg/ml). Cells were cultured in a humidified atmosphere of 5% CO2 at 37◦ C. The MDA-MB-231 cells were incubated in Leibovitz’s L-15 medium with 15% heat-inactivated fetal calf serum (FCS), L-glutamine (2 mM) and gentamycin (50 μg/ml) at 37◦ C in humidified conditions without CO2 . The presence of leptin was search in serum and culture media by ELISA (Human Leptin ELISA Kit, RayBiotech, Norcross, GA, sensitivity = 2 pg/mL) and was not detectable. Immunohistochemistry The expression of leptin and Ob-Rt was investigated by immunohistochemical staining using affinity-purified goat polyclonal biotinylated antibodies against leptin and Ob-Rt (recognizing the different isoforms of Ob-R; R&D, Abingdon, UK). Cells were grown on plastic slides for 48 h in a 37◦ C humidified atmosphere with 5% CO2 before fixing with acetone for 10 min. Nonspecific binding sites were blocked using the avidin/biotin kit for 30 min (Abcys, Paris, France). Slides were then incubated overnight at 4◦ C in a humidified chamber with the antileptin and anti-Ob-Rt biotinylated antibodies (1 μg/ml). Endogenous peroxidase activity was inhibited with 0.3% hydrogen peroxide for 5 min. Visualization was carried out using a Vectastain ABC peroxidase-conjugated streptavidine kit for 30 min (Abcys, Paris, France). The sections were then treated with DAB substrate for 10 min to give staining. Finally, slides were contrasted using haematoxylin, dehydrated and mounted using the Vectastain mounting medium (Abcys, Paris, France). For each assay, control samples without the antileptin or antiOb-Rt antibody or without the peroxidase revelation kit were used to establish the specificity of the immunohistochemical analysis. Cell Proliferation Assay Resazurin Assay Cells were propagated in 96-well plates with complete media for 24 h. Cells were then washed with phosphate buffered saline (PBS) and exposed to physiological (10 ng/ml), obese (100 ng/ml), or pharmacologic (1,000 ng/ml) concentrations of human recombinant leptin (R&D, Abingdon, UK) in a medium supplemented with or without FCS or HS for 24, 48, 72, and

647

96 h without replacing the medium. After washing with PBS, 200 μl of a 25 μg/ml solution of resazurin in medium was added to each well. Plates were incubated 2 h at 37◦ C in a humidified atmosphere containing 5% CO2 . Fluorescence was then measured on an automated 96-well plate reader (Fluoroskan Ascent FL, Thermo Fisher Scientific, Wilmington, DE) using an excitation wavelength of 530 nm and an emission wavelength of 590 nm. Under these conditions, fluorescence (OD value) was proportional to the number of living cells in the well (37). Cell proliferation assay was performed at least 3 times for each cell line (in replicates of 6 wells for each concentration). xCELLigence Assay Cell proliferation and/or survival was also monitored through xCELLigence RTCA system (Roche Diagnosis, Meylan, France), which allows label-free monitoring changes of cell phenotype, that is, cell number, viability, morphology and quality of cell attachment by measure of cell-to electrode responses of cells seeded in E-96-well plates manufactured with integrated microelectronic sensor arrays. Cell index or cell index normalized at time of treatment were calculated for each E-plate well by RTCA Software 1.2 (Roche Diagnosis, Meylan, France). Data are represented by time-dependent mean cell indexes. Apoptosis Apoptosis was evaluated using the Annexin V-FITC kit (Beckman Coulter, Roissy, France). Cells were plated (165,000 cells) in 6-well plates in complete medium and incubated at 37◦ C in a humidified atmosphere containing 5% CO2 . After 24 h, cells were exposed to fresh medium without FCS (increases spontaneous apoptosis) containing increasing concentrations of leptin (10, 100, and 1,000 ng/ml). After 72 h of treatment, cells were harvested with trypsin and 10% FCS in PBS was added as soon as the cells were released from the dish. Then 3 × 105 cells were washed with PBS and incubated 10 min with Annexin VFITC and propidium iodide (PI) according to the manufacturer’s protocol. Cells were analysed on a Coulter Epics XL flow cytometry system (Beckman Coulter, Roissy, France), placing the FITC signal in FL1 and the PI signal in FL4. Intact cells were gated in the FSC/SSC plot to exclude small debris. Cells in the lower right quadrant of the FL1/FL4 dot plot (labelled with Annexin V-FITC only) were considered to be in early apoptosis, and cells in the upper right quadrant (labeled with Annexin V-FITC and PI) were in late apoptosis/necrosis. Cell Cycle Modification of cell cycle was analyzed by flow cytometry. Cells were plated (165,000 cells) in 6-well plates in complete medium and incubated at 37◦ C in a humidified atmosphere containing 5% CO2 . After 24 h, cells were exposed to fresh medium without FCS containing different concentrations of leptin (10, 100, and 1000 ng/ml). After 72 h of treatment, cells were harvested with trypsin and 10% FCS in PBS was added as soon as the cells were released from the dish. Then 3 × 105 cells

648

V. DUBOIS ET AL.

Downloaded by [Memorial University of Newfoundland] at 00:55 26 January 2015

were washed with PBS and incubated 30 min with RNAse and PI according to the manufacturer’s protocol. Cells were FACSanalyzed as previously described, placing the PI signal in FL4. RNA Extraction and Reverse Transcription Cells were propagated in 96-well plates with complete media for 24 h. Cells were washed with PBS and exposed to leptin (100 and 1000 ng/ml) for 96 h (n = 4 for each cell line) in a medium without HS or FCS. Total cellular RNA were extracted with Trizol reagent according to the manufacturer instructions (Invitrogen, Carlsbad, CA). The purity and integrity of the RNA were validated using a NanoDrop 8000 spectrophotometer (Thermo Fisher Scientific, Wilmington, DE) and by electrophoresis in 2% agarose gel. Reverse transcription was conducted from 1 μg of total RNA, giving rise to 20 μl of cDNA as previously described (38). Quantitative RT-PCR The RT-synthesised cDNAs were amplified using primers summarized in Table 1. The quantitative PCR was realized using Light Cycler-FastStart DNA master SYBR Green I according to the manufacturer manual on a Light Cycler Instrument (Roche Diagnostics, Mannheim, Germany). Sample mRNA copy numbers were extrapolated from standard curves obtained with serially diluted purified PCR products. Relative levels of gene expression were calculated by target gene levels normalized to the endogenously expressed 18S gene. The second derivative maximum method was used to determine the Crossing point (Cp) for each sample (realized in triplicate). The mean value was used to calculate the Cp (Cp of the target gene minus Cp of the 18S gene). Expression levels of genes were obtained according to the transformation (2−Cp). To confirm the identity of the PCR products, the generated bands were sequenced on both strands, with the same primers described above used in the amplification and the DNA dye terminator cycle sequencing kit (Applied Biosytems, Courtaboeuf,

France). Sequence analysis was performed with an Applied Biosystems model 377 DNA Sequencer. Western Blot Cells were plated (165,000 cells) in 6-well plates in complete medium and incubated at 37◦ C in a humidified atmosphere containing 5% CO2 . After 24 h, cells were exposed to fresh medium without FCS containing increasing concentrations of leptin (10, 100, and 1000 ng/ml). After 72 h of treatment, cells were harvested with trypsin and 10% FCS in PBS was added as soon as the cells were released from the dish. Cell lysates, obtained with “RIPA-like” (containing 10μg of total proteins), were separated on 4–12% SDS PAGE gels (Invitrogen, Carlsbad, CA), transferred to nitrocellulose and blotted using various antibodies [p21 (1/200), Bax (1/200) and c-myc (1/1,000)]. Secondary horseradish peroxidase conjugated antibodies were obtained from Santa Cruz (antirabbit) or Dako (antimouse). Immunoreactive bands were visualized by incubation with DURA Western blotting detection system from Thermo-Scientific (Waltham, MA). Glyceraldehyde 3-phosphate dehydrogenase (1/20,000) monoclonal antibody was used as a loading control. Statistical Analysis Statistical analysis was performed using paired Student’s t-test. Differences with P < 0.1 were considered to be subsignificant and with P < 0.05, statistically significant compared to control. RESULTS Expression of Leptin and Leptin Receptor in Breast Cells Expressions of leptin and Ob-R were analyzed using 2 complementary methods: the quantitative RT-PCR and the immunohistochemistry. Leptin, Ob-Rt, and Ob-Rl mRNA expression was detected in the normal breast cell line (184B5; data not shown), in human fibrocystic cell line (MCF10A), and in

TABLE 1 Primers used in quantitative RT-PCR Gene

Primer

Sequence

Leptin

Forward Reverse Forward Reverse Forward Reverse Forward Reverse Forward Reverse Forward Reverse

5’-TGAGCACCTGCTTCATGCTC-3’ 5’-TGAGTGCGGTTTGACCACTG-3’ 5’-CATTTTATCCCCATTGAGAAGTA-3’ 5’-CTGAAAATTAAGTCCTTGTGCCCAG-3’ 5’-GATAGAGGCCCAGGCATTTTTTA-3’ 5’-ACACCACTCTCTCTCTTTTTGATTGA-3’ 5’-GTGTACAACTACCCCGAGGGC-3’ 5’-AAACCCCCCAGGCCGTTGGAG-3’ 5’-CAAGGTTATTTTGATGCATGG-3’ 5’-AATCCTTGACAGACTTCTCAT-3’ 5’-GTCTGTGATGCCCTTAGATG-3’ 5’-AGCTTATGACCCGCACTTAC-3’

OB-Rt Ob-Rl Estrogen receptor Aromatase 18S

LEPTIN PROLIFERATIVE RESPONSE IN BREAST CANCER CELLS

649

Downloaded by [Memorial University of Newfoundland] at 00:55 26 January 2015

As expected, medium with FCS or HS induced significantly exponential proliferation in the four breast cell lines (Supplemental Fig. S1). Without HS, the proliferation of MCF10A cells was limited (Supplemental Fig. S1). In T47D cells, medium without FCS stopped the proliferation from 48 h. In contrast, treatment without FCS in MCF-7 and MDA-MB-231 lines decreased significantly the number of cells since 24 h.

FIG. 1. mRNA and protein expression of leptin and leptin receptors in untreated normal breast cell line (MCF10A) and in untreated breast cancer cells (MCF-7, MDA-MB-231, and T47D). A: The mRNA expression of leptin, the long isoform of leptin receptor (Ob-Rl) and isoforms of leptin receptor (Ob-Rt) was analyzed by quantitative RT-PCR. B: Leptin and Ob-Rt proteins were detected in breast cell lines using immunohistochemistry. Magnification, ×400. (Color figure available online.)

3 human cancer cell lines (MCF-7, MDA-MB-231 and T47D; Fig. 1A). In addition, the expression levels of leptin, Ob-Rt, and Ob-Rl mRNA were similar in breast cancer cells and in fibrocystic cell line (P > 0.01). Consistent with the expression of mRNAs, leptin, and Ob-Rt proteins were detected in all cell lines (Fig. 1B). The leptin and Ob-Rt proteins were localized both in the cytoplasm and nucleus of cells. Effect of Serum on Breast Cell Proliferation MCF10A fibrocystic breast cells and MCF-7, MDA-MB231, and T47D breast cancer cells were cultured using medium supplemented with or without FCS or HS. 184B5 breast cell were cultured with serum-supplemented media. The aim of the experiment was to assess the effect of leptin alone or in association with multiple growth factors contained in FCS or HS on cell proliferation.

Effects of Leptin on Breast Cell Proliferation 184B5 normal cells, MCF10A fibrocystic cells, and MCF-7, MDA-MB-231, T47D cancer cells were treated with increasing concentrations of leptin corresponding to human normal circulating levels (10 ng/ml), obesity-associated levels (100 ng/ml), and pharmacologic concentrations (1000 ng/ml) for 96 h. On 184B5 normal breast cells, leptin had no significant effect on cell proliferation neither by fluorescent analysis (Supplemental Fig. S2A) nor by kinetic measurement with xCELLigence (Supplemental Fig. S2B). Regarding MCF10A cell line, using a medium supplemented with FCS, leptin (1000 ng/ml) enhanced significantly the proliferation by 15.6% and 15.2% at 72 and 96 h, respectively (Fig. 2A). At 100 ng/ml, the stimulatory effect was significant at 96 h (5.7%). Without FCS, the stimulatory effect of leptin (1000 ng/ml) was stronger by significantly increasing the proliferation to 21.1%, 36.0% and 45.5% at 48, 72, and 96 h, respectively (Figure 2B). At 100 ng/ml, the effect of leptin was less pronounced (+13.7 at 72 h, P < 0.1; +10.6% at 96 h, P < 0.05). Under the 2 different culture conditions (FCS-supplemented or -unsupplemented), leptin treatment did not modify the cell proliferation of MDA-MB-231 cells whatever time, dose, and technique used (data not shown). On T47D, using a medium supplemented with FCS, leptin (1000 ng/ml) induced a significant proliferative response to 5.5%, 7.4%, and 10% at 48, 72, and 96 h, respectively (Fig. 2C). At 100 ng/ml, the stimulatory effect was subsignificant at 72 h and significant at 96 h. Without FCS, cells were sensitive to low leptin levels since leptin once 10 ng/ml enhanced the proliferation at 48 h (7.3%) and 72 h (14.5%) and limited the cell death at 96 h (+34.4%) (Fig. 2D). On MCF-7, using a medium supplemented with FCS, the maximal stimulatory effect was observed for leptin concentration at 1,000 ng/ml (Figure 2E). Leptin presented a subsignificant stimulation to 5.1% at 48 h and a significant proliferative response at 72 (+9.7%) and 96 h (+14.6%). Using a lower concentration of leptin (100 ng/ml), the stimulatory effect was less pronounced (+5.9% at 72 h, P < 0.1; +6.4% at 96 h, P < 0.05). Without FCS, leptin (1000 ng/ml) induced a subsignificant response at 24 h (+29.8%) and a significant effect at 48 (+50.4%), 72 (+86.2%) and, 96 h (+134%) (Fig. 2F). In this model, the number of control cells decreased with time. Interestingly, leptin at 1000 ng/ml maintained the survival of

650

V. DUBOIS ET AL.

MCF10A 200

A

HS+ *

*

HS-

B

OD value

150 Control

*

100

10 100

* 50

*

1000

Leptin (ng/ml)

*

0 24

48

72

96

24

48

72

96

T ime (hours)

T47D 200

FCS+

FCS-

C

D

OD value

150

* 100

*

Control 10

*

*

*

50

*

*

***

***

100 1000

Leptin (ng/ml)

0 24

48

72

96

24

48

72

96

T ime (hours)

MCF7 200

FCS+

FCS-

E

F

150

OD value

Downloaded by [Memorial University of Newfoundland] at 00:55 26 January 2015

*

*

*

* Control 10

100

100 1000

Leptin (ng/ml)

50

**

*

*

*

*

0 24

48

72

96

24

48

72

96

T ime (hours)

FIG. 2. Influence of leptin on cells. Cells were cultured in 96-well plates with complete medium for 24 h. The MCF10A were then exposed to 3 concentrations of leptin in media supplemented with (A) or without (B) horse serum for the times indicated. The T47D were then exposed to three concentrations of leptin in media supplemented with (C) or without (D) fetal calf serum for the times indicated. The relative number of viable cells was estimated using the resazurin assay. The relative number of MCF7 viable cells was estimated after exposition to 3 concentrations of leptin in media supplemented with (E) or without (F,G) fetal calf serum for the times indicated using the resazurin assay (E,F) and Xcelligence method (G). Bars give means + SEM obtained from 1 to 6 determinations. Significant differences from controls are flagged as ∗ P < 0.05. (Continued)

Downloaded by [Memorial University of Newfoundland] at 00:55 26 January 2015

LEPTIN PROLIFERATIVE RESPONSE IN BREAST CANCER CELLS

651

FIG. 2. (Continued).

serum-deprived cancer cells because no significant difference was observed between different times. The lower dose (100 ng/ml) of leptin gave a limited response by inducing a significant response to 23.6%, 36.0%, and 38.3% at 48, 72, and 96 h, respectively. Using xCELLigence method, also realized without FCS, leptin also induced the survival of MCF7 cells (Fig, 2G). Effects of Leptin on Apoptosis and Cell Cycle Because leptin did not alter the proliferation of 184B5 and MDA-MB-231 cells, the effects of leptin on apoptosis, cell cycle and signaling molecules were not analyzed on these cells. After 72 h of treatment with recombinant human leptin at 100 and 1000 ng/ml, the proportion of MCF7 cells in apoptotic stage significantly decreased by 19% and 16% respectively (Fig. 3A), but the cell cycle was not altered (Fig. 3B). Leptin did not significantly modify apoptosis or cell cycle of MCF10A and T47D cells at 72 h (data not shown). After 72 h of treatment, treatment with leptin, expression of Bax, C-myc, and p21 proteins were not altered (Fig. 3C). We specifically selected these factors because they are known regulators of cell proliferation and apoptosis. Bax is a proapoptotic factor, p21 is a negative regulator of cell cycle progression, and c-myc is a master regulator of both cell proliferation and apoptosis. Effects of Leptin on Leptin Pathway The effects of leptin were evaluated using the most effective model (medium without FCS) and the most efficient concentrations (100 and 1000 ng/ml) and time (96 h) for leptin treatment. Because leptin was inefficient on 184B5 and MDA-MB-231 cell proliferation, the effect of leptin on 184B5 and MDA-MB-231 leptin pathway was not analyzed. On MCF10A cells, leptin did not modify the expression of leptin, Ob-Rt, and Ob-Rl mRNA (Fig. 4). On MCF-7 cells, leptin (1000 ng/ml) significantly enhanced the expression of leptin (+45.5%) and Ob-Rl (+109.5%) mRNA (Fig. 4). Considering Ob-Rt mRNA, a subsignificant stimulatory effect was observed (+25.9%).

On T47D cells, leptin at 1000 ng/ml induced a significant positive response on Ob-Rt mRNA expression (+34%) and a subsignificant effect on Ob-Rl mRNA (+55.7%) (Fig. 4). Effects of Leptin on Estrogen Pathway MCF-7 and T47D cells were exposed to leptin (100 and 1000 ng/ml) for 96 h and mRNA levels of aromatase and ERα were analyzed using quantitative RT-PCR. Because MCF10A and MDA-MB-231 are ERα negative, we did not investigate the effect of leptin on ERα expression. In addition, these 2 cell lines did not express aromatase (38). On MCF-7, leptin (1,000 ng/ml) induced a subsignificant positive response on aromatase mRNA expression (+41.7%) (Fig. 5). In addition, leptin at 100 ng/ml significantly stimulated the expression of ERα (+75.6%) but was inefficient at 1000 ng/ml. About T47D, the expression of aromatase mRNA was sub-significantly enhanced (+66.5%) by leptin at 1,000 ng/ml (Fig. 5). DISCUSSION Obesity is a risk factor for breast cancer in postmenopausal women. Obese subjects are characterized by metabolic disorders and notably hyperleptinemia. Several lines of evidence suggested that leptin, mainly synthesized by adipose tissue, may be involved in breast cancer development. We observed that leptin and Ob-R mRNA as well as proteins were expressed in MCF-7, MDA-MB-231, and T47D breast cancer cells, in accordance with numerous previous studies (28,32,40,41). We also described these expressions in 184B5 normal cells and MCF10A cells, a noncancerous fibrocystic cell line. These results are in agreement with our previous studies conducted on human biopsies showing leptin and Ob-R protein expression in breast cancer cells and in benign breast lesions (18,42). Moreover, in the present study, we did not observed differences in leptin, Ob-Rt, and Ob-Rl mRNA expression levels between breast fibrocystic and cancer cells. These results may suggest that leptin is implicated locally in breast benign or malignant cell regulation acting via an autocrine and/or paracrine pathway. Interestingly, leptin may play a role on its own

Downloaded by [Memorial University of Newfoundland] at 00:55 26 January 2015

652

V. DUBOIS ET AL.

FIG. 3. Effect of leptin on the apoptosis (A), cell cycle (B), and on the expression of protein involved in apoptosis and cell cycle of breast cancer cells. MCF7 breast cancer cells were cultured in 6-well plates with complete medium for 24 h and were then exposed to leptin (10, 100, and 1000 ng/ml) during 72 h. Bars give means + SEM obtained from 4 determinations. Significant differences from controls are flagged as ∗ P < 0.05. (Color figure available online.)

pathway. Indeed, we observed that high leptin levels (1000 ng/ml) were able to induce an overexpression of Ob-Rt and Ob-Rl mRNA in MCF-7 and T47D cancer cells. Moreover, the expression of leptin mRNA was enhanced by leptin treatment in MCF-7 cells. Similar results were obtained in ZR-75-1 breast cancer cells using low leptin levels (100 ng/ml) (28). Another study noted that leptin treatment induced protein expression of Ob-Rl but was inefficient on leptin and Ob-Rt in MCF-7 cells (32). These results are particularly important in severe obesity as increased circulating leptin levels may lead to

overexpression of leptin and Ob-R in breast cancer cells, hence producing a vicious loop. In addition, this feedback process in cancer cells may explain why human breast cancer tissues are suggested to overexpress leptin and Ob-R (43,44). We conducted experiments to characterize the effects of leptin on breast cell proliferation. We used 2 complementary techniques: the resazurin-based assay that allows the measurement of cell proliferation at specific time-points and the xCELLigence system that monitors cell growth in real-time. Similar results were obtained using these 2 techniques. The use of medium supplemented with or without HS or FCS allowed us to analyze the effects of leptin on different systems: (a) a survival assay, in which cell proliferation decreased with time (without FCS or HS) and (b) a highly proliferative systems in which cells undergo exponential proliferation (with FCS or HS). In the present study, in a medium supplemented with HS or FCS, leptin is still able to enhance proliferation even in the presence of multiple growth factors. Without FCS, leptin maintained the survival of MCF-7 cells during 96 h, suggesting that leptin, at high dose, was susceptible to induce a survival signal through cell cycle induction and/or apoptosis inhibition. Indeed, leptin at high dose decreased global apoptosis at 72 h, in accordance with previous results (45). Nevertheless, cell cycle was not changed by leptin in MCF7 cells. We did not observe a dose-dependent effect on cell cycle or apoptosis. Early time points could have been used. Using a 72-h time-point, which is where there is a significant difference in terms of cell number, we are maybe missing early leptin-associated mechanisms that lead to increase in cell number. Furthermore, we previously reported using phenol red-free conditions (46), leptin (at 1000ng/ml) significantly increased the proliferation to +13.4% and 16.3% at 72 and 96 h, respectively, in MCF7 cells. In the present study, using media containing phenol red, a similar response was observed following leptin treatment (+9.7% at 72 h; +14.6 at 96 h). These results suggest that the proliferative response of leptin is not mediated by a potential phenol red-associated estrogenic response. We noted that leptin, using obese plasma concentrations (100 ng/ml) or pharmacological doses (1000 ng/ml), stimulated also MCF10A fibrocystic mammary cell growth and T47D breast cancer cell proliferation. These observations are in accordance with previous results obtained on MCF-7 (27,40,47,48), MDA-MB-361 (31), T47D (27,49,50) breast cancer cells. For the first time we showed that leptin had no effect on the proliferation of the normal cell line 184B5, in contrast with the results obtained with the fibrocystic mammary cell line MCF10A. Leptin was also inefficient on MDA-MB-231 breast cancer cells, in contrast with previous studies showing proliferative activity of leptin (32,34). This discrepancy may be explained by different methodology, including culture conditions and cell viability assay. Fusco et al. showed that leptin-induced MCF7 cell proliferation was associated with an increased expression of ERα receptor and this response was not present in the MDA-MB 231 cells. Moreover in this study, the effects induced by leptin on MCF7 cells were abolished when Ob-R

653

LEPTIN PROLIFERATIVE RESPONSE IN BREAST CANCER CELLS

MCF-7

300

Relative mRNA level

Relative mRNA level

MCF10A 250 200 150 100 50 0 Leptin

Ob-Rt

300

*

250 200

Control 100 Leptin 1000 (ng/ml)

*

150 100 50 0

Leptin

Ob-Rl

Ob-Rt

Ob-Rl

Relative mRNA level

300 250 200

*

150 100 50 0 Leptin

Ob-Rt

Ob-Rl

FIG. 4. Effect of leptin on the expression of leptin, Ob-Rt, and Ob-Rl mRNA in breast cells. MCF10A fibrocystic breast cells and MCF-7 and T47D breast cancer cells were cultured in 96-well plates with complete medium for 24 h and were then exposed to leptin (100 and 1000 ng/ml) during 96 h in media without horse serum or fetal calf serum. Results were expressed as mean normalized relative to the control + SEM obtained from 4 determinations. Significant differences from controls are flagged as ∗ P < 0.05.

was neutralized using either a monoclonal inhibitory antibody raised against Ob-R or Ob-R gene silencing siRNA (31). Interestingly, in the present study, leptin was efficient on ER-positive breast cancer cell lines (MCF-7 and T47D) but ineffective on ERα-negative MDA-MB-231 breast cancer cells, suggesting a possible relationship between leptin and estrogen pathway. Previously, we noted a positive correlation between Ob-R and ER expression using immunohistochemical approach on breast cancer biopsies (18). In this way, we analyzed in vitro the effect of leptin on mRNA expression of aromatase and ERα in MCF-7 and T47D cells. Leptin induced a subsignificant overexpression of aromatase mRNA in these 2 breast cancer cell lines. This finding is consistent with the results from Catalano et al. that demonstrated leptin-positive-enhancement of aromatase mRNA expression, aromatase protein content, and its enzymatic activity in MCF-7 cells (35). However, a recent study did not observe such effect of leptin on the aromatase protein content in

MCF-7

MCF-7 and T47D (32). We also observed overexpression of ERα induced by leptin (100 ng/ml) in MCF-7 cancer cells. This suggested a potential relationship between leptin and estrogen pathways. These results seem particularly important in obese women with hyperleptinemia because leptin may induce the in situ production of estrogens via the stimulation of aromatase production and may enhance the sensibility of breast cells to estrogens via the upregulation of ERα. Given the limited number of studies providing data on the cross-talk between leptin and estrogen signaling pathways, further experiments are required to clearly demonstrate and understand their relationship (51,52). However, because ERα negative MCF-10A cells are leptin-sensitive, leptin-induced cell proliferation cannot be explained only by estrogen-dependent mechanisms. In conclusion, we observed that fibrocystic and cancer cells expressed leptin and Ob-R. Leptin treatment induced upregulation of leptin, Ob-Rt, and Ob-Rl mRNA in cancer cells.

T47D

250

*

200 150 100 50 0 Aromatase

Estrogen receptor α

Relative mRNA level

Relative mRNA level

Downloaded by [Memorial University of Newfoundland] at 00:55 26 January 2015

T47D

250 200

Control 100 Leptin 1000 (ng/ml)

150 100 50 0 Aromatase

Estrogen receptor α

FIG. 5. Effect of leptin on the expression of aromatase and estrogen receptor α mRNA in breast cancer cells. MCF-7 and T47D cells were cultured in 96-well plates with complete medium for 24 h and were then exposed to leptin (100 and 1000 ng/ml) during 96 h in medium without fetal calf serum. Results were expressed as mean normalized relative to the control + SEM obtained from 4 determinations. Significant differences from controls are flagged as ∗ P < 0.05.

654

V. DUBOIS ET AL.

Downloaded by [Memorial University of Newfoundland] at 00:55 26 January 2015

These results suggest a positive leptin signaling, inducing local leptin production and leptin-sensitivity of cancer cells via over-production of Ob-R. In addition, we showed that leptin stimulated the proliferation of fibrocystic and cancer cells, but not normal breast cells. Finally, we noted a potential relationship between leptin and estrogen, leptin treatment inducing a probable expression of aromatase and expression of ERα. The positive autoregulation induced by leptin, its proliferative and antiapoptotic activities, and its relationship with estrogen pathway in breast cancer cells may explain that obesity, via hyperleptinemia, can be considered as a risk factor for breast cancer development. SUPPLEMENTAL DATA Supplemental data for this article can be accessed on the publisher’s website. ACKNOWLEGEMENTS We are indebted to C. Forestier (Laboratoire de Bact´eriologie, UFR Pharmacie, Clermont-Ferrand, France) for expert technical assistance. Virginie Dubois and Thierry Jard´e contributed equally to this work. FUNDING This work received financial support from the French Anti-Cancer League office (Ligue Contre le Cancer du Puyde-Dˆome) and “Canc´eropˆole Lyon-Auvergne-Rhˆone-Alpes.” Virginie Dubois and Thierry Jard´e were supported by a fellowship from the French Ministry of Research and Technology. REFERENCES 1. Bergstrom A, Pisani P, Tenet V, Wolk A, and Adami HO: Overweight as an avoidable cause of cancer in Europe. Int J Cancer 91, 421–430, 2001. 2. Bianchini F, Kaaks R, and Vainio H: Overweight, obesity, and cancer risk. Lancet Oncol 3, 565–574, 2002. 3. Maccio A, Madeddu, and Mantovani G: Adipose tissue as target organ in the treatment of hormone-dependent breast cancer: new therapeutic perspectives. Obes Rev 10, 660–670, 2009. 4. Daling, JR, Malone KE, Doody DR, Johnson LG, Gralow JR, et al.: Relation of body mass index to tumor markers and survival among young women with invasive ductal breast carcinoma. Cancer 92, 720–729, 2001. 5. Irigaray P, Newby JA, Lacomme S, and Belpomme D: Overweight/obesity and cancer genesis: more than a biological link. Biomed Pharmacother 61, 665–678, 2007. 6. Key TJ, Allen NE, Verkasalo PK, and Banks E: Energy balance and cancer: the role of sex hormones. Proc Nutr Soc, 60, 81–89, 2001. 7. Galic S, Oakhill JS, and Steinberg GR: Adipose tissue as an endocrine organ. Mol Cell Endocrinol 316, 129–139, 2010. 8. Somasundar P, McFadden DW, Hileman SM, and Vona-Davis L: Leptin is a growth factor in cancer. J Surg Res 116, 337–349, 2004. 9. Liuzzi A, Savia G, Tagliaferri M, Lucantoni R, Berselli ME, et al.: Serum leptin concentration in moderate and severe obesity: relationship with clinical, anthropometric and metabolic factors. Int J Obes Relat Metab Disord 23, 1066–1073, 1999.

10. Clement K, Vaisse C, Lahlou N, Cabrol S, Pelloux V, et al.: A mutation in the human leptin receptor gene causes obesity and pituitary dysfunction. Nature 392, 398–401, 1998. 11. Hancke K, Grubeck D, Hauser N, Kreienberg R, Weiss JM, et al.: Adipocyte fatty acid-binding protein as a novel prognostic factor in obese breast cancer patients. Breast Cancer Res Treat 119, 367–370, 2010. 12. Tessitore., Vizio B, Pesola D, Cecchini F, Mussa A, et al.: Adipocyte expression and circulating levels of leptin increase in both gynaecological and breast cancer patients. Int J Oncol 24, 1529–1535, 2004. 13. Hou, WK, Xu YX, Yu T, Zhang L, Zhang WW, et al.: Adipocytokines and breast cancer risk. Chin Med J (Engl) 120, 1592–1596, 2007. 14. Ozet A, Arpaci F, Yilmaz MI, Ayta H, Ozturk B, et al.: Effects of tamoxifen on the serum leptin level in patients with breast cancer. Jpn J Clin Oncol 31, 424–427, 2001. 15. Han CZ, Du LL, Jing JX, Zhao XW, Tian FG, et al.: Associations among lipids, leptin, and leptin receptor gene Gin223Arg polymorphisms and breast cancer in China. Biol Trace Elem Res 126, 38–48, 2008. 16. Carroll PA, Healy L, Lysaght J, Boyle T, Reynolds JV, et al.: Influence of the metabolic syndrome on leptin and leptin receptor in breast cancer. Mol Carcinog 50, 643–651, 2011. 17. Chen DC, Chung YF, Yeh YT, Chaung HC, Kuo FC, et al.: Serum adiponectin and leptin levels in Taiwanese breast cancer patients. Cancer Lett 237, 109–114, 2006. 18. Jarde T, Caldefie-Ch´ezet F, Damez M, Mishellany F, Penault-Llorca F, et al.: Leptin and leptin receptor involvement in cancer development: a study on human primary breast carcinoma. Oncol Rep 19, 905–911, 2008. 19. Jeong YJ, Bong JG, Park SH, Choi JH, and Oh HK: Expression of leptin, leptin receptor, adiponectin, and adiponectin receptor in ductal carcinoma in situ and invasive breast cancer. J Breast Cancer 14, 96–103, 2012. 20. Miyoshi Y, Funahashi T, Tanaka S, Taguchi T, Tamaki Y, et al., High expression of leptin receptor mRNA in breast cancer tissue predicts poor prognosis for patients with high, but not low, serum leptin levels. Int J Cancer 118, 1414–1419, 2006. 21. Cleary MP, Phillips FC, Getzin SC, Jacobson TL, Jacobson MK, et al.: Genetically obese MMTV-TGF-alpha/Lep(ob)Lep(ob) female mice do not develop mammary tumors. Breast Cancer Res Treat 77, 205–215, 2003. 22. Cleary MP, Juneja SC, Phillips FC, Hu X, Grande JP, et al.: Leptin receptordeficient MMTV-TGF-alpha/Lepr(db)Lepr(db) female mice do not develop oncogene-induced mammary tumors. Exp Biol Med (Maywood) 229, 182–193, 2004. 23. Zheng Q, Dunlap SM, Zhu J, Downs-Kelly E, Rich J, et al.: Leptin deficiency suppresses MMTV-Wnt-1 mammary tumor growth in obese mice and abrogates tumor initiating cell survival. Endocr Relat Cancer 18, 491–503, 2011. 24. Mauro L, Catalano S, Bossi G, Pellegrino M, Barone I, et al.: Evidences that leptin up-regulates E-cadherin expression in breast cancer: effects on tumor growth and progression. Cancer Res 67, 3412–3421, 2007. 25. Gonzalez RR, et al.: Leptin signaling promotes the growth of mammary tumors and increases the expression of vascular endothelial growth factor (VEGF) and its receptor type two (VEGF-R2). J Biol Chem 281, 26320–26328, 2006. 26. Gonzalez-Perez RR, Cherfils S, Escobar M, Yoo JH, Carino C, et al.: Leptin upregulates VEGF in breast cancer via canonic and non-canonical signalling pathways and NFkappaB/HIF-1alpha activation. Cell Signal 22, 1350–1362, 2010. 27. Dieudonne MN, Machinal-Quelin F, Serazin-Leroy V, Leneveu MC, Pecquery R, et al.: Leptin mediates a proliferative response in human MCF7 breast cancer cells. Biochem Biophys Res Commun 293, 622–628, 2002. 28. Soma D, Kitayama J, Yamashita H, Miyato H, Ishikawa M, et al.: Leptin augments proliferation of breast cancer cells via transactivation of HER2. J Surg Res 149, 9–14, 2008. 29. Yin N, Wang D, Zhang H, Yi X, Sun X, et al.: Molecular mechanisms involved in the growth stimulation of breast cancer cells by leptin. Cancer Res 64, 5870–5855, 2004.

Downloaded by [Memorial University of Newfoundland] at 00:55 26 January 2015

LEPTIN PROLIFERATIVE RESPONSE IN BREAST CANCER CELLS 30. Nkhata KJ, Ray A, Schuster TF, Grossmann ME, and Cleary MP: Effects of adiponectin and leptin co-treatment on human breast cancer cell growth. Oncol Rep 21, 1611–1619, 2009. 31. Fusco R, Galgani M, Procaccini C, Franco R, Pirozzi G, et al.: Cellular and molecular crosstalk between leptin receptor and estrogen receptor-{alpha} in breast cancer: molecular basis for a novel therapeutic setting. Endocr Relat Cancer 17, 373–382, 2010. 32. Ray A, Nkhata KJ, and Cleary MP: Effects of leptin on human breast cancer cell lines in relationship to estrogen receptor and HER2 status. Int J Oncol 30, 1499–1509, 2007. 33. McCormack D, Schneider J, McDonald D, and McFadden D: The antiproliferative effects of pterostilbene on breast cancer in vitro are via inhibition of constitutive and leptin-induced Janus kinase/signal transducer and activator of transcription activation. Am J Surg 202, 541–544, 2011. 34. Otvos L Jr, Kovalszky I, Riolfi M, Ferla R, Olah J, et al.: Efficacity of leptin receptor antagonist peptide in a mouse model of triple-negative breast cancer. Eur J Cancer 47, 1578–1584, 2011. 35. Catalano S, Marsico S, Giordano C, Mauro L, Rizza P, et al.: Leptin enhances, via AP-1, expression of aromatase in the MCF-7 cell line. J Biol Chem 278, 28668–28676, 2003. 36. Catalano S, Mauro L, Marsico S, Giordano C, Rizza P, 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. 37. Debiton E, Madelmont JC, Legault J, and Barthomeuf C: Sanguinarineinduced apoptosis is associated with an early and severe cellular glutathione depletion. Cancer Chemother Pharmacol 51, 474–482, 2003. 38. Goncalves-Mendes N, Blanchon L, Meiniel A, Dastugue B, and Sapin V: Placental expression of SCO-spondin during mouse and human development. Gene Expr Patterns 4, 309–314, 2004. 39. Yang C, Yu B, Zhou D, and Chen S: Regulation of aromatase promoter activity in human breast tissue by nuclear receptors. Oncogene 21, 2854–2863, 2002. 40. 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 upregulation of cyclin D1 and c-Myc and down-regulation of tumor suppressor p53 and p21WAF1/CIP1. Breast Cancer Res Treat 98, 121–132, 2006.

655

41. 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. 42. Caldefie-Chezet F., Damez M, de Latour M, Konska G, Mishellani F, et al.: Leptin: a proliferative factor for breast cancer? Study on human ductal carcinoma. Biochem Biophys Res Commun 334, 737–741, 2005. 43. Garofalo C, Koda M, Cascio S, Sulkowska M, Kanczuga-Koda L, 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. 44. 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. 45. Jiang H, Yu J, Guo H, Song H, and Chen S: Upregulation of survivin by leptin/STAT3 signaling in MCF-7 cells. Biochem Biophys Res Commun 368, 1–5, 2008. 46. Jarde T, Caldefie-Ch´ezet F, Goncalves-Mendes N, Mishellany F, Buechler C, et al., Involvement of adiponectin and leptin in breast cancer: clinical and in vitro studies. Endocr Relat Cancer 16, 1197–1210, 2009. 47. Okumura M, Yamamoto M, Sakuma H, Kojima T, Maruyama T, et al.: Leptin and high glucose stimulate cell proliferation in MCF-7 human breast cancer cells: reciprocal involvement of PKC-alpha and PPAR expression. Biochim Biophys Acta 1592, 107–116, 2002. 48. 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. 49. Hu X, Juneja SC, Maihle NJ, and Cleary MP: Leptin—a growth factor in normal and malignant breast cells and for normal mammary gland development. J Natl Cancer Inst 94, 1704–1711, 2002. 50. 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. 51. Jarde T, Perrier S, Vasson MP and Caldefie-Ch´ezet F: Molecular mechanisms of leptin and adiponectin in breast cancer. Eur J Cancer 47, 33–43, 2011. 52. Khandekar MJ, Cohen P, and Spiegelman, BM: Molecular mechanisms of cancer development in obesity. Nat Rev Cancer 11, 886–895, 2011.

Leptin induces a proliferative response in breast cancer cells but not in normal breast cells.

Obesity is a risk factor for breast cancer in postmenopausal women. Leptin, a hormone excessively produced during obesity, is suggested to be involved...
554KB Sizes 0 Downloads 3 Views