Cell Biochem Biophys DOI 10.1007/s12013-014-0047-9
ORIGINAL PAPER
Adiponectin Induces Breast Cancer Cell Migration and Growth Factor Expression Zhongming Jia • Yan Liu • Shouyong Cui
Ó Springer Science+Business Media New York 2014
Abstract Adiponectin, the hormone produced and secreted by adipocytes, has been shown to promote migration of the epithelial cells and angiogenesis in these cells. We sought to determine if adiponectin could induce the cellular migration and growth factor expression in breast cancer cells grown in vitro. The breast cancer cell lines MDA-MB-436 and MFM-223 (estrogen-independent) were treated with adiponectin for different time periods. Supernatants of the cell cultures were obtained by centrifugation and were assayed for growth factor expression by the enzyme-linked immunosorbent assay (ELISA). Becton–Dickinson-Falcon Transwell systems were used to assay adiponectin-induced migration. Adiponectin significantly induced the expression of various growth factors, including vascular endothelial growth factor, transforming growth factor-b1, and basic fibroblast growth factor in MDA-MB-436 and MFM-223 cells. Adiponectin also enhanced the migration of breast cancer cells which were inhibited about 50–70 % by the inhibitors of mitogenactivated protein kinase and phosphatidylinositol 3-kinase
Zhongming Jia and Yan Liu equally contributed to this work. Z. Jia (&) Department of Thyroid and Breast Surgery, Affiliated Hospital of Binzhou Medical College, Binzhou 256610, People’s Republic of China e-mail: [email protected] Y. Liu Department of Gynecology, Binzhou People’s Hospital, Binzhou 256610, People’s Republic of China S. Cui Department of General Emergency Surgery, Affiliated Hospital of Binzhou Medical College, Binzhou 256610, People’s Republic of China
(PI3K). Adiponectin treatment of the cancer cell induced an increased expression of different growth factors and migration of the cells. These effects are likely to contribute to the progression of breast cancer, implying that change in adiponectin levels associated with obesity may be considered as a high risk factor in breast cancer patients. Keywords
Adiponectin Migration Breast cancer
Introduction Obesity, a worldwide problem approaching epidemic proportions, has been established as a risk factor for several pathophysiological conditions including heart diseases, hypertension, diabetes, and many forms of cancer [1, 2]. The implication of excess weight in cancer caused mortality has also been recognized suggesting that obese patients of various forms of cancer are at a higher risk of death [3]. According to a study, about 14 % of the cancer-related deaths in the past decades were attributed to obesity [3]. Several types of cancers including those of esophagus, colon, breast, endometrium, pancreas, and gall bladder have been found to be affected by obesity [1–4]. An understanding of the association of excess weight with cancers, particularly of the underlying mechanisms will certainly assist in developing preventive therapeutic strategies. Various cytokines and/or hormones produced and secreted by the adipose tissue including adiponectin have been envisaged to play a role in the promotion of breast cancer [5, 6]. Particularly, in the postmenopausal and ERpositive breast cancer, adiponectin has been suggested to play an independent role [7]. Adiponectin, is a peptide hormone exclusively synthesized and secreted by the adipocytes and its plasma levels in
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the overweight people are found to be lower than the lean individuals [8]. The postmenopausal women have been shown to exhibit significantly higher levels of adiponectin than the premenopausal women [7]. Several studies have indicated that low levels of circulating adiponectin are associated with many forms of cancer including that of the breast. That is, the plasma levels of adiponectin are inversely related to the incidence of a malignancy [9]. Furthermore the link of obesity associated lower levels of adiponectin with breast cancer has been deemed to be independent of other causative factors, such as hormone status, age, menopausal status, and estrogen receptors [8]. Serum levels of adiponectin secreted from mature adipocytes have been shown to be inversely related to the breast cancer [10, 11]. Various growth factors and cytokines are known to promote the growth, and metastasis of breast tumors and induce angiogenesis. Thus, increased levels of vascular endothelial growth factor (VEGF), basic fibroblast growth factor (bFGF), transforming growth factor-b1 (TGF-b1), interleukin-8, interleukin-6, and tumor necrosis factor-a have been shown to promote the proliferation of breast cancer cells. The in vitro grown breast cancers have been shown to inversely respond to the adiponectin concentrations, that is, the increased levels of this cytokine caused a decline in the cancer cell proliferation [12]. In fact, increased levels of several obesity-linked growth factors and endogenous sex steroids contribute to the breast cancer progression [13]. Understanding of the mechanisms involved in the obesity-malignancy link will certainly assist to improve the preventive and therapeutic measures of breast cancer. It has been shown that change in adiponectin levels impacts the proliferation and migration of endothelial and epithelial cells, and is capable of promoting angiogenesis. In this study, we have evidenced that adiponectin induced an increased expression of the growth factors that control the cell proliferation. We also evidenced adiponectin-induced stimulation of the cell migration indicative of metastasis in the breast cancer cells grown in vitro.
Adiponectin Treatments and Growth Factor Expression MDA-MB-436 or MFM-223 cells were plated (1 9 106 cells per well) of a 6-well plate and allowed to grow overnight. Cells were then serum-deprived for 20 h followed by treatment with 0, 4, 40, or 80 ng/mL of human recombinant adiponectin (R&D Systems, Minneapolis, MN, USA) for 0, 0.5, 1, 2, 4, 24, or 48 h. Levels of secreted VEGF, TGF-b1, bFGF, and epidermal growth factor (EGF) from cell supernatants were quantified using an enzyme linked immunosorbent assay (ELISA) (R&D Systems). Each point of the data represent an average of 3 concentrations (pg/mL) measured in individual well. Cell Migration MDA-MB-436 or MFM-223 cell lines were seeded (2 9 105 cell/mL) into the upper chamber of a BD-Falcon Transwell system (12-well format; 8 lM/L pore size) in a serum-free medium and allowed to grow overnight. Migration was induced by adding 0, 4, 40, or 80 ng/mL adiponectin to the lower chamber. For inhibition studies, MAPK inhibitor (U0126; 10 or 20 lM/L) or PI3 K inhibitor (LY294002; 20 or 40 lM/L) was added to the upper and lower chambers (Promega, Madison, WI, USA). After 24 h, membranes were fixed and stained with methylene blue. The migrated cells were counted (3 fields/membrane) in three separately conducted experiments and an average was determined. Statistical Analysis Determination of statistical significance was performed by analysis of variance. Comparison of individual concentration means with the control was completed using the Tukey–Kramer multiple comparison test. Data are reported as means ± standard errors. Results
Methods
Expression of VEGF
Cell Lines
The expression of VEGF in MDA-MB-436 (Fig. 1a) and MFM-223 (Fig. 1b) cells grown in the absence and presence of adiponectin was determined. In MDA-MB-436 cells, the adiponectin treatment produced a time-dependent and significant (p \ 0.0001) increase in VEGF levels; however, the magnitude of increase was equal at all the doses of adiponectin. The VEGF levels in MDA-MB-436 cells treated for 48 h exceeded the detection range of the ELISA. In MFM-223 cells, a time-dependent and significant (p \ 0.0001) increase, noticed at a minimum of 2 h treatment, is evident; however, adiponectin appeared to produce no effect.
The cell lines were maintained at 37 °C in a 5 % CO2 atmosphere. MFM-223 and MDA-MB-436 are 2 estrogenindependent breast cancer cell lines (American Type Culture Collection, Manassas, VA, USA). MFM-223 cells were maintained in F12 K media (CellGro, Herndon, VA, USA), and MDA-MB-436 cells were maintained in RPMI 1640 media (BioWhittaker, Walkersville, MD, USA). The media were supplemented with 10 % fetal bovine serum (Gibco, Rockville, MD, USA) and 1 % antibiotics (100 U/ mL penicillin, 100 mg/mL streptomycin; BioWhittaker).
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Cell Biochem Biophys Fig. 1 Effect of adiponectin on the expression of VEGF in MDA-MB-436 (a) and MFM223 (b) cells; The cells were grown in the presence of 0 (filled circle with dash), 4 (open rectangle), 40 (open square), or 80 (times) ng/mL adiponectin for 0, 0.5, 1, 2, 4, 24, and 48 h. At the end of the incubation, the supernatants were assayed for VEGF expression by ELISA. The values of VEGF expressed as pg/ml represent an average of three observations. The effect was considered significant when the value of p was \0.05
Fig. 2 Expression of TGF-b1 in MDA-MB-436 (a) and MFM223 (b) cells in response to varying concentrations of adiponectin; Cells were treated with 0 (filled circle with dash), 4 (open rectangle), 40 (open square), or 80 (times) ng/mL of adiponectin for 0 or 0.5–48 h. Then the supernatants were assayed for TGF-b1 expression by ELISA. The values of TGFb1expressed in pg/mL represent an average of three independent estimations. Effect considered significant when the p value was \0.05
Expression of TGF-b1 Expression of TGF-b1 was determined in MDA-MB-436 (Fig. 2a) and MFM-223 (Fig. 2b) cells grown in the absence
and presence of adiponectin. In both these cells, with all the doses of adiponectin, a significant and uniform increase (p \ 0.0001) in the levels of TGF-b1 was observed. The increase was first noticed at 4 h of the treatment time and
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Cell Biochem Biophys Fig. 3 Effect of adiponectin on expression of bFGF in MDAMB-436 (a) and MFM-223 (b); the cells were treated with 0 (filled circle with dash), 4 (open rectangle), 40 (open square), or 80 (times) ng/mL adiponectin for 0.5–48 h. Then the cell supernatants were assayed for bFGF expression in triplicate by ELISA. The values of bfGF expressed in pg/mL represent an average of three determinations. Compared to the control, the effect was considered significant if the value of p was \0.05
then continued to elevate further with increasing time of incubation.
adiponectin did produce an augmentation of the bFGF response, however, with no well-defined trend.
Expression of EGF and bFGF
Adiponectin-Induced Cell Migration
MDA-MB-436 and MFM-223 cells showed no expression of EGF either in the presence or absence of adiponectin (data not shown). The bFGF levels expressed in MDA-MB436 (Fig. 3a) and MFM-223 (Fig. 3b) cells were also low. In MDA-MB-436 cells, they were negligible at 0.5 and 1 h, increased significantly by 2–4 h and declined to 0 by 24 h. A substantial increase after 48 h of incubation was detected. In response to adiponectin, the bFGF was significantly (p \ 0.0001) and dose dependently increased at 2 h. However, 4 and 40 lg/mL of adiponectin produced exactly the same amount of increase. Following the pattern of control, the response to adiponectin was reduced at 4 h of incubation though it still showed augmentation. Then by 24 h, response to all concentrations of adiponectin was reduced to 0. The bFGF generation was increased substantially again in response to 4 lg/mL of adiponectin and showed an augmentation compared to the control. However, the higher concentrations of adiponectin significantly reduced the bFGF generation. In MFM-223 cells, the bFGF was detected at the 0.5 h and it was not increased with the increasing time of incubation. Addition of adiponectin caused an augmentation which was higher with 40 lg/mL of adiponectin and lower with 4 and 80 lg/mL. In brief, the
Migration of MDA-MB-436 and MFM-223 cells (Fig. 4a) was determined in response to increasing concentrations of adiponectin. The augmentation of migration was dosedependent as it increased with the increasing concentrations of adiponectin. At a concentration of 80 ng/mL, adiponectin increased the number of migrated MFM-223 and MDA-MB-436 cells by 2.3-fold (p \ 0.05) and 1.8fold, respectively. The movement of cells caused by 40 ng/ mL of adiponectin was inhibited by inhibitors of MAPK (LY294002) and PI3 K (U0126) (Fig. 4b). The MAPK inhibitor, LY294002 at a concentration of 40 lM, inhibited the adiponectin-augmented migration of the MFM-223 cells by 50 % and this effect was significant (p \ 0.05). The migration of MDA-MB-436 cells was inhibited by 70 % with 20 lM/L U0126 (p \ 0.05).
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Discussion The MDA-MB-436 and MFM-223 cell lines are known to grow independent of estrogen and develop metastatic tumors in the nude mice [14, 15]. Previously, the breast cancer cells have been shown to express adiponectin
Cell Biochem Biophys
Fig. 4 a Effects of adiponectin on migration of MDA-MB-436 and MFM-223 cells in response to 0, 4, 40, or 80 ng/mL of adiponectin treatment for 24 h; The number of migrated cells was normalized to the number obtained with 0 ng/mL adiponectin as control. * p \ 0.05. b Effects of the inhibitors of MAPK U0126 and PI3 K LY294002 on adiponectin-augmented migration in MDA-MB-436 and MFM-223 cells. Cells were exposed to 40 ng/mL adiponectin, with or without
LY294002 or U0126, and the number of migrated cells was determined after 24 h. Data were normalized to the values obtained with 40 ng/mL adiponectin as control. * p \ 0.05. The data are representative of three independently conducted experiments. Three fields per membrane were counted to determine the average number of migrated cells per experiment. Gray bars MFM-223 cells, black bars MDA-MB-436 cells
receptors, and therefore they are capable of responding to adiponectin [16]. In this study, our objective was to determine the role of exogenously added adiponectin on the expression of growth factors and migration of the breast cancer cells, MDA-MB-436 and MFM-223. The growth factor VEGF is a potent mitogen that is capable of stimulating cellular migration, angiogenesis, and microvascular permeability. An over expression of VEGF has been associated with a large number of human tumors and it has been shown to correlate with tumor stage, grade, and clinical outcome in breast cancer. In fact, VEGF has been suggested to be a molecular link between lymphangiogenesis and metastasis [17, 18]. Adiponectin has been found to retard the growth of implanted tumor, neovascularization, angiogenesis, and VEGF expression and therefore suggested to reduce the malignancy by directly regulating different signaling pathways related to tumor growth [18]. Contrarily, our results also showed a significant increase in VEGF levels in response to adiponectin in MDA-MB-436 but not in MFM-223 cells. Thus, adiponectin treatment of the MDA-MB-436 cells for 24 h resulted in doubling of the secreted VEGF. In agreement with these observations, adiponectin has been shown to
induce VEGF in human and mouse macrophages through ERK signaling pathway [19]. Another group evidenced that adiponectin was pro-inflammatory in the mouse colon cancer, and it induced different growth and inflammation promoting cytokines and growth factors by activating adiponectin receptors in the primary epithelial cells with malignant phenotype [20]. Certainly, this report also supports our results. Whereas an increased expression and activity of TGFb1 have been associated with obesity, this growth factor has also been evidenced to inhibit adipogenesis [21]. The increased messenger RNA production of TGF-b1 has been detected in both benign and malignant breast cancer cells. Furthermore, TGF-b1 levels have been found to increase metastasis and invasiveness of the cancer cells. A direct correlation between TGF-b1 and the circulating levels of breast cancer-specific antigen are also reported [22]. We determined the effect of adiponectin on TGF-b1 expression in MFM-223 and MDA-MB-436 cells. Interestingly, we found that treatment of these cell lines with adiponectin, caused a significant augmentation of the TGF-b1 expression in a time-dependent manner. A treatment of MDAMB-436 and MFM-223 cells with adiponectin for 48 h led
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to an average of 3- and 2-fold increase in TGF-b1 expression, respectively, indicating a pro-carcinogenic role of adiponectin. The increased circulating levels of bFGFhave been detected in breast cancer patients. This growth factor functions to maintain the integrity of epithelial cells and thereby protects against inflammation. Adiponectin has been shown to exert part of its pro-inflammatory action by binding with bFGF and thereby blocking its protective activity [20]. In this study, we found low expression of bFGF in MDA-MB-436 cells; however, when these cells were treated with adiponectin for 2 or 4 h, bFGF generation was significantly increased. Surprisingly, the bFGF expression was declined to 0 with 24 h of adiponectin treatment. In MFM-223 cells, however, the adiponectininduced increase remained more or less unchanged from 0.5 to 48 h of incubation. It is hard to derive a definitive conclusion from these observations as it is likely that due to binding of adiponectin with bFGF in the supernatants of cell culture interfered with the accurate detection. Another mitogenic growth factor EGF and over expression of its receptors has led to intensive research in developing drugs for blocking this carcinogenic activity [23]. However, in MDA-MB-436 and MFM-223 cells, we detected no expression of EGF either in the presence or absence of adiponectin. As discussed earlier, the vascularization in breast cancer is correlated directly with metastasis and cell invasion. Our data also showed that adiponectin-induced in vitro motility of breast cancer cells was substantially blocked by the inhibitors of MAPK and PI3 K. Clearly, these findings suggest that adiponectin promotes metastasis and invasion of cancer cells through MAPK and PI3 K signaling pathways. In summary, our results indicate that of adiponectin, dose dependently increased the in vitro motility of breast cancer cells, and it also augmented the expression of mitogenic and angiogenic growth factors, VEGF and TGFb1 in these cells.Certainly, these results do not explain as to why the obesity characterized by low circulating levels of adiponectin is a higher risk for cancer. Also, contrary to many reports, the mitogenic effects of adiponectin presently obtained suggest that further studies are needed to understand the role of adiponectin in linking obesity to breast cancer. The protective role of adiponectin against breast cancer was hypothesized and high levels of circulating adiponectin were associated with decreased risk of breast cancer first by Miyoshi et al. [24]. While this finding was supported by several of the subsequent retrospective case–control studies [7, 25, 26], many other investigations reported contradictory results suggesting that adiponectin was not strongly related to the risk of breast cancer [27]. Then, it was also
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indicated that blood levels of adiponectin were not associated with breast cancer independent of other known risk factors [28]. The inconsistency in the levels of female sex hormones, menstrual status etc. have been ascribed to the lack of a well defined association between adiponectin and breast cancer [29]. Several reports indicated that low levels of adiponectin are a high risk factor for breast cancer in postmenopausal but not in the premenopausal women [25– 27, 30]. A study based on meta-analysis including results of many investigations, indicated of inconsistency and therefore a difficulty in ascertaining if the effect of adiponectin on cancer promotion is negative or positive [31]. Our results further support that circulating adiponectin levels may not always be used as negative risk factor for breast cancer and related complications. It has also been shown that using a different antibody for immunoblotting, very little reduction in adiponectin expression related to stimulated adipogenesis was observed [22]. This finding raises the question as to whether the techniques used for the detection of adiponectin concentrations provide with an accurate estimation. Further studies are needed to define the role of adiponectin in promoting or suppressing the cancer-related signaling and its relation to hormone status, gender, age, and weight of the patient.
References 1. van Kruijsdijk, R., van der Wall, E., & Visseren, F. (2009). Obesity and cancer: The role of dysfunctional adipose tissue. Cancer Epidemiology, Biomarkers and Prevention, 18(10), 2569–2578. 2. Schlienger, J. L., et al. (2009). Obesity and cancer. Revue de Medecine Interne, 30(9), 776–782. 3. Calle, E. E., et al. (2003). Overweight, obesity, and mortality from cancer in a prospectively studied cohort of U.S. adults. New England Journal of Medicine, 348(17), 1625–1638. 4. Neugut, A. I., Chen, A. C., & Petrylak, D. P. (2004). The ‘‘skinny’’ on obesity and prostate cancer prognosis. Journal of Clinical Oncology, 22(3), 395–398. 5. Dalamaga, M., Diakopoulos, K. N., & Mantzoros, C. S. (2012). The role of adiponectin in cancer: A review of current evidence. Endocrine Reviews, 33(4), 547–594. 6. Kelesidis, I., Kelesidis, T., & Mantzoros, C. (2006). Adiponectin and cancer: A systematic review. British Journal of Cancer, 94(9), 1221–1225. 7. Tian, Y. F., et al. (2007). Anthropometric measures, plasma adiponectin, and breast cancer risk. Endocrine-Related Cancer, 14(3), 669–677. 8. Cnop, M., et al. (2003). Relationship of adiponectin to body fat distribution, insulin sensitivity and plasma lipoproteins: evidence for independent roles of age and sex. Diabetologia, 46, 459–469. 9. Perrier, S., & Jarde, T. (2012). Adiponectin, an anti-carcinogenic hormone? A systematic review on breast, colorectal, liver and prostate cancer. Current Medicinal Chemistry, 19(32), 5501–5512. 10. Wu, M. H., et al. (2009). Circulating levels of leptin, adiposity and breast cancer risk. British Journal of Cancer, 100(4), 578–582.
Cell Biochem Biophys 11. Sun, C. A., et al. (2010). Adipocytokine resisting and breast cancer risk. Breast Cancer Research and Treatment, 123(3), 869–876. 12. Grossmann, M. E., et al. (2008). Balance of adiponectin and leptin modulates breast cancer cell growth. Cell Research, 18(11), 1154–115625. 13. Prieto-Hontoria, P. L., et al. (2011). Role of obesity-associated dysfunctional adipose tissue in cancer: A molecular nutrition approach. Biochimica Et Biophysica Acta-Bioenergetics, 1807(6SI), 664–678. 14. Zhang, X. T., et al. (2012). Estrogen receptor-alpha 36 mediates mitogenic antiestrogen signaling in ER-negative breast cancer cells. PLoS One, 7(1), e30174. doi:10.1371/journal.pone. 0030174. 15. Grossmann, M. E., et al. (2008). Effects of adiponectin on breast cancer cell growth and signaling. British Journal of Cancer, 98, p370–p379. 16. Ko¨rner, A., et al. (2007). Total and high molecular weight adiponectin in breast cancer: In vitro and in vivo studies. J Clin Endocrinol & Metabolism, 92(3), 1041–1048. 17. Lijnen, H. R., & Scroyen, I. (2013). Effect of vascular endothelial growth factor receptor 2 antagonism on adiposity in obese mice. Journal of Molecular Endocrinology, 50(3), 319–324. 18. Moon, H. S., et al. (2013). Salutary effects of adiponectin on colon cancer: In vivo and in vitro studies in mice. Gut, 62(4), 561–570. 19. Hu, D., et al. (2013). Adiponectin regulates vascular endothelial growth factor-C expression in macrophages via Syk-ERK pathway. PLOS One, 8, e560712. 20. Fayad, R., et al. (2007). Adiponectin deficiency protects mice from chemically induced colonic inflammation. Gastroenterology, 132(2), 601–614. 21. Du, B. W., et al. (2013). The transcription factor paired-related homeobox 1 (Prrx1) inhibits adipogenesis by activating
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transforming growth factor-beta (TGF beta) signaling. Journal of Biological Chemistry, 288(5), 3036–3047. Wanninger, J., et al. (2011). Adiponectin induces the transforming growth factor decoy receptor BAMBI in human hepatocytes. FEBS Letters, 585(9), 1338–1344. Masuda, H., et al. (2012). Role of Epidermal Growth Factor Receptor in Breast Cancer. Breast Cancer Research and Treatment,. doi:10.1007/s10549-012-2289-9. Miyoshi, Y., et al. (2003). Association of serum adiponectin levels with breast cancer risk. Clinical Cancer Research, 9, 5699–5704. Hou, W. K., et al. (2007). Adipocytokines and breast cancer risk. Chinese Medical Journal (English Edition), 120, 1592–1596. Kang, J. H., et al. (2007). Relationship of serum adiponectin and resistin levels with breast cancer risk. Journal of Korean Medical Science, 22, 117–121. doi:10.3346/jkms.2007.22.1.117. Mantzoros, C., et al. (2004). Adiponectin and breast cancer risk. Journal of Clinical Endocrinology and Metabolism, 89, 1102–1107. doi:10.1210/jc.2003-031804. Tworoger, S. S., et al. (2007). Plasma adiponectin concentrations and risk of incident breast cancer. Journal of Clinical Endocrinology and Metabolism, 92, 1510–1516. doi:10.1210/jc.20061975. Gaudet, M. M., et al. (2010). Do adipokines underlie the association between known risk factors and breast cancer among a cohort of United States women. Cancer Epidemiology, 34, 580–586. doi:10.1016/j.canep.2010.05.014. Miyatani, Y., et al. (2008). Associations of circulating adiponectin with estradiol and monocyte chemotactic protein-1 in postmenopausal women. Menopause, 15, 536–541. doi:10.1097/ gme.0b013e31815c85ed. Liu, L.-Y., et al. (2013). The role of adiponectin in breast cancer: A meta-analysis. PloS One, 8(8), e73183. doi:10.1371/journal. pone.0073183.
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ORIGINAL PAPER
Adiponectin Induces Breast Cancer Cell Migration and Growth Factor Expression Zhongming Jia • Yan Liu • Shouyong Cui
Ó Springer Science+Business Media New York 2014
Abstract Adiponectin, the hormone produced and secreted by adipocytes, has been shown to promote migration of the epithelial cells and angiogenesis in these cells. We sought to determine if adiponectin could induce the cellular migration and growth factor expression in breast cancer cells grown in vitro. The breast cancer cell lines MDA-MB-436 and MFM-223 (estrogen-independent) were treated with adiponectin for different time periods. Supernatants of the cell cultures were obtained by centrifugation and were assayed for growth factor expression by the enzyme-linked immunosorbent assay (ELISA). Becton–Dickinson-Falcon Transwell systems were used to assay adiponectin-induced migration. Adiponectin significantly induced the expression of various growth factors, including vascular endothelial growth factor, transforming growth factor-b1, and basic fibroblast growth factor in MDA-MB-436 and MFM-223 cells. Adiponectin also enhanced the migration of breast cancer cells which were inhibited about 50–70 % by the inhibitors of mitogenactivated protein kinase and phosphatidylinositol 3-kinase
Zhongming Jia and Yan Liu equally contributed to this work. Z. Jia (&) Department of Thyroid and Breast Surgery, Affiliated Hospital of Binzhou Medical College, Binzhou 256610, People’s Republic of China e-mail: [email protected] Y. Liu Department of Gynecology, Binzhou People’s Hospital, Binzhou 256610, People’s Republic of China S. Cui Department of General Emergency Surgery, Affiliated Hospital of Binzhou Medical College, Binzhou 256610, People’s Republic of China
(PI3K). Adiponectin treatment of the cancer cell induced an increased expression of different growth factors and migration of the cells. These effects are likely to contribute to the progression of breast cancer, implying that change in adiponectin levels associated with obesity may be considered as a high risk factor in breast cancer patients. Keywords
Adiponectin Migration Breast cancer
Introduction Obesity, a worldwide problem approaching epidemic proportions, has been established as a risk factor for several pathophysiological conditions including heart diseases, hypertension, diabetes, and many forms of cancer [1, 2]. The implication of excess weight in cancer caused mortality has also been recognized suggesting that obese patients of various forms of cancer are at a higher risk of death [3]. According to a study, about 14 % of the cancer-related deaths in the past decades were attributed to obesity [3]. Several types of cancers including those of esophagus, colon, breast, endometrium, pancreas, and gall bladder have been found to be affected by obesity [1–4]. An understanding of the association of excess weight with cancers, particularly of the underlying mechanisms will certainly assist in developing preventive therapeutic strategies. Various cytokines and/or hormones produced and secreted by the adipose tissue including adiponectin have been envisaged to play a role in the promotion of breast cancer [5, 6]. Particularly, in the postmenopausal and ERpositive breast cancer, adiponectin has been suggested to play an independent role [7]. Adiponectin, is a peptide hormone exclusively synthesized and secreted by the adipocytes and its plasma levels in
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Cell Biochem Biophys
the overweight people are found to be lower than the lean individuals [8]. The postmenopausal women have been shown to exhibit significantly higher levels of adiponectin than the premenopausal women [7]. Several studies have indicated that low levels of circulating adiponectin are associated with many forms of cancer including that of the breast. That is, the plasma levels of adiponectin are inversely related to the incidence of a malignancy [9]. Furthermore the link of obesity associated lower levels of adiponectin with breast cancer has been deemed to be independent of other causative factors, such as hormone status, age, menopausal status, and estrogen receptors [8]. Serum levels of adiponectin secreted from mature adipocytes have been shown to be inversely related to the breast cancer [10, 11]. Various growth factors and cytokines are known to promote the growth, and metastasis of breast tumors and induce angiogenesis. Thus, increased levels of vascular endothelial growth factor (VEGF), basic fibroblast growth factor (bFGF), transforming growth factor-b1 (TGF-b1), interleukin-8, interleukin-6, and tumor necrosis factor-a have been shown to promote the proliferation of breast cancer cells. The in vitro grown breast cancers have been shown to inversely respond to the adiponectin concentrations, that is, the increased levels of this cytokine caused a decline in the cancer cell proliferation [12]. In fact, increased levels of several obesity-linked growth factors and endogenous sex steroids contribute to the breast cancer progression [13]. Understanding of the mechanisms involved in the obesity-malignancy link will certainly assist to improve the preventive and therapeutic measures of breast cancer. It has been shown that change in adiponectin levels impacts the proliferation and migration of endothelial and epithelial cells, and is capable of promoting angiogenesis. In this study, we have evidenced that adiponectin induced an increased expression of the growth factors that control the cell proliferation. We also evidenced adiponectin-induced stimulation of the cell migration indicative of metastasis in the breast cancer cells grown in vitro.
Adiponectin Treatments and Growth Factor Expression MDA-MB-436 or MFM-223 cells were plated (1 9 106 cells per well) of a 6-well plate and allowed to grow overnight. Cells were then serum-deprived for 20 h followed by treatment with 0, 4, 40, or 80 ng/mL of human recombinant adiponectin (R&D Systems, Minneapolis, MN, USA) for 0, 0.5, 1, 2, 4, 24, or 48 h. Levels of secreted VEGF, TGF-b1, bFGF, and epidermal growth factor (EGF) from cell supernatants were quantified using an enzyme linked immunosorbent assay (ELISA) (R&D Systems). Each point of the data represent an average of 3 concentrations (pg/mL) measured in individual well. Cell Migration MDA-MB-436 or MFM-223 cell lines were seeded (2 9 105 cell/mL) into the upper chamber of a BD-Falcon Transwell system (12-well format; 8 lM/L pore size) in a serum-free medium and allowed to grow overnight. Migration was induced by adding 0, 4, 40, or 80 ng/mL adiponectin to the lower chamber. For inhibition studies, MAPK inhibitor (U0126; 10 or 20 lM/L) or PI3 K inhibitor (LY294002; 20 or 40 lM/L) was added to the upper and lower chambers (Promega, Madison, WI, USA). After 24 h, membranes were fixed and stained with methylene blue. The migrated cells were counted (3 fields/membrane) in three separately conducted experiments and an average was determined. Statistical Analysis Determination of statistical significance was performed by analysis of variance. Comparison of individual concentration means with the control was completed using the Tukey–Kramer multiple comparison test. Data are reported as means ± standard errors. Results
Methods
Expression of VEGF
Cell Lines
The expression of VEGF in MDA-MB-436 (Fig. 1a) and MFM-223 (Fig. 1b) cells grown in the absence and presence of adiponectin was determined. In MDA-MB-436 cells, the adiponectin treatment produced a time-dependent and significant (p \ 0.0001) increase in VEGF levels; however, the magnitude of increase was equal at all the doses of adiponectin. The VEGF levels in MDA-MB-436 cells treated for 48 h exceeded the detection range of the ELISA. In MFM-223 cells, a time-dependent and significant (p \ 0.0001) increase, noticed at a minimum of 2 h treatment, is evident; however, adiponectin appeared to produce no effect.
The cell lines were maintained at 37 °C in a 5 % CO2 atmosphere. MFM-223 and MDA-MB-436 are 2 estrogenindependent breast cancer cell lines (American Type Culture Collection, Manassas, VA, USA). MFM-223 cells were maintained in F12 K media (CellGro, Herndon, VA, USA), and MDA-MB-436 cells were maintained in RPMI 1640 media (BioWhittaker, Walkersville, MD, USA). The media were supplemented with 10 % fetal bovine serum (Gibco, Rockville, MD, USA) and 1 % antibiotics (100 U/ mL penicillin, 100 mg/mL streptomycin; BioWhittaker).
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Cell Biochem Biophys Fig. 1 Effect of adiponectin on the expression of VEGF in MDA-MB-436 (a) and MFM223 (b) cells; The cells were grown in the presence of 0 (filled circle with dash), 4 (open rectangle), 40 (open square), or 80 (times) ng/mL adiponectin for 0, 0.5, 1, 2, 4, 24, and 48 h. At the end of the incubation, the supernatants were assayed for VEGF expression by ELISA. The values of VEGF expressed as pg/ml represent an average of three observations. The effect was considered significant when the value of p was \0.05
Fig. 2 Expression of TGF-b1 in MDA-MB-436 (a) and MFM223 (b) cells in response to varying concentrations of adiponectin; Cells were treated with 0 (filled circle with dash), 4 (open rectangle), 40 (open square), or 80 (times) ng/mL of adiponectin for 0 or 0.5–48 h. Then the supernatants were assayed for TGF-b1 expression by ELISA. The values of TGFb1expressed in pg/mL represent an average of three independent estimations. Effect considered significant when the p value was \0.05
Expression of TGF-b1 Expression of TGF-b1 was determined in MDA-MB-436 (Fig. 2a) and MFM-223 (Fig. 2b) cells grown in the absence
and presence of adiponectin. In both these cells, with all the doses of adiponectin, a significant and uniform increase (p \ 0.0001) in the levels of TGF-b1 was observed. The increase was first noticed at 4 h of the treatment time and
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Cell Biochem Biophys Fig. 3 Effect of adiponectin on expression of bFGF in MDAMB-436 (a) and MFM-223 (b); the cells were treated with 0 (filled circle with dash), 4 (open rectangle), 40 (open square), or 80 (times) ng/mL adiponectin for 0.5–48 h. Then the cell supernatants were assayed for bFGF expression in triplicate by ELISA. The values of bfGF expressed in pg/mL represent an average of three determinations. Compared to the control, the effect was considered significant if the value of p was \0.05
then continued to elevate further with increasing time of incubation.
adiponectin did produce an augmentation of the bFGF response, however, with no well-defined trend.
Expression of EGF and bFGF
Adiponectin-Induced Cell Migration
MDA-MB-436 and MFM-223 cells showed no expression of EGF either in the presence or absence of adiponectin (data not shown). The bFGF levels expressed in MDA-MB436 (Fig. 3a) and MFM-223 (Fig. 3b) cells were also low. In MDA-MB-436 cells, they were negligible at 0.5 and 1 h, increased significantly by 2–4 h and declined to 0 by 24 h. A substantial increase after 48 h of incubation was detected. In response to adiponectin, the bFGF was significantly (p \ 0.0001) and dose dependently increased at 2 h. However, 4 and 40 lg/mL of adiponectin produced exactly the same amount of increase. Following the pattern of control, the response to adiponectin was reduced at 4 h of incubation though it still showed augmentation. Then by 24 h, response to all concentrations of adiponectin was reduced to 0. The bFGF generation was increased substantially again in response to 4 lg/mL of adiponectin and showed an augmentation compared to the control. However, the higher concentrations of adiponectin significantly reduced the bFGF generation. In MFM-223 cells, the bFGF was detected at the 0.5 h and it was not increased with the increasing time of incubation. Addition of adiponectin caused an augmentation which was higher with 40 lg/mL of adiponectin and lower with 4 and 80 lg/mL. In brief, the
Migration of MDA-MB-436 and MFM-223 cells (Fig. 4a) was determined in response to increasing concentrations of adiponectin. The augmentation of migration was dosedependent as it increased with the increasing concentrations of adiponectin. At a concentration of 80 ng/mL, adiponectin increased the number of migrated MFM-223 and MDA-MB-436 cells by 2.3-fold (p \ 0.05) and 1.8fold, respectively. The movement of cells caused by 40 ng/ mL of adiponectin was inhibited by inhibitors of MAPK (LY294002) and PI3 K (U0126) (Fig. 4b). The MAPK inhibitor, LY294002 at a concentration of 40 lM, inhibited the adiponectin-augmented migration of the MFM-223 cells by 50 % and this effect was significant (p \ 0.05). The migration of MDA-MB-436 cells was inhibited by 70 % with 20 lM/L U0126 (p \ 0.05).
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Discussion The MDA-MB-436 and MFM-223 cell lines are known to grow independent of estrogen and develop metastatic tumors in the nude mice [14, 15]. Previously, the breast cancer cells have been shown to express adiponectin
Cell Biochem Biophys
Fig. 4 a Effects of adiponectin on migration of MDA-MB-436 and MFM-223 cells in response to 0, 4, 40, or 80 ng/mL of adiponectin treatment for 24 h; The number of migrated cells was normalized to the number obtained with 0 ng/mL adiponectin as control. * p \ 0.05. b Effects of the inhibitors of MAPK U0126 and PI3 K LY294002 on adiponectin-augmented migration in MDA-MB-436 and MFM-223 cells. Cells were exposed to 40 ng/mL adiponectin, with or without
LY294002 or U0126, and the number of migrated cells was determined after 24 h. Data were normalized to the values obtained with 40 ng/mL adiponectin as control. * p \ 0.05. The data are representative of three independently conducted experiments. Three fields per membrane were counted to determine the average number of migrated cells per experiment. Gray bars MFM-223 cells, black bars MDA-MB-436 cells
receptors, and therefore they are capable of responding to adiponectin [16]. In this study, our objective was to determine the role of exogenously added adiponectin on the expression of growth factors and migration of the breast cancer cells, MDA-MB-436 and MFM-223. The growth factor VEGF is a potent mitogen that is capable of stimulating cellular migration, angiogenesis, and microvascular permeability. An over expression of VEGF has been associated with a large number of human tumors and it has been shown to correlate with tumor stage, grade, and clinical outcome in breast cancer. In fact, VEGF has been suggested to be a molecular link between lymphangiogenesis and metastasis [17, 18]. Adiponectin has been found to retard the growth of implanted tumor, neovascularization, angiogenesis, and VEGF expression and therefore suggested to reduce the malignancy by directly regulating different signaling pathways related to tumor growth [18]. Contrarily, our results also showed a significant increase in VEGF levels in response to adiponectin in MDA-MB-436 but not in MFM-223 cells. Thus, adiponectin treatment of the MDA-MB-436 cells for 24 h resulted in doubling of the secreted VEGF. In agreement with these observations, adiponectin has been shown to
induce VEGF in human and mouse macrophages through ERK signaling pathway [19]. Another group evidenced that adiponectin was pro-inflammatory in the mouse colon cancer, and it induced different growth and inflammation promoting cytokines and growth factors by activating adiponectin receptors in the primary epithelial cells with malignant phenotype [20]. Certainly, this report also supports our results. Whereas an increased expression and activity of TGFb1 have been associated with obesity, this growth factor has also been evidenced to inhibit adipogenesis [21]. The increased messenger RNA production of TGF-b1 has been detected in both benign and malignant breast cancer cells. Furthermore, TGF-b1 levels have been found to increase metastasis and invasiveness of the cancer cells. A direct correlation between TGF-b1 and the circulating levels of breast cancer-specific antigen are also reported [22]. We determined the effect of adiponectin on TGF-b1 expression in MFM-223 and MDA-MB-436 cells. Interestingly, we found that treatment of these cell lines with adiponectin, caused a significant augmentation of the TGF-b1 expression in a time-dependent manner. A treatment of MDAMB-436 and MFM-223 cells with adiponectin for 48 h led
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Cell Biochem Biophys
to an average of 3- and 2-fold increase in TGF-b1 expression, respectively, indicating a pro-carcinogenic role of adiponectin. The increased circulating levels of bFGFhave been detected in breast cancer patients. This growth factor functions to maintain the integrity of epithelial cells and thereby protects against inflammation. Adiponectin has been shown to exert part of its pro-inflammatory action by binding with bFGF and thereby blocking its protective activity [20]. In this study, we found low expression of bFGF in MDA-MB-436 cells; however, when these cells were treated with adiponectin for 2 or 4 h, bFGF generation was significantly increased. Surprisingly, the bFGF expression was declined to 0 with 24 h of adiponectin treatment. In MFM-223 cells, however, the adiponectininduced increase remained more or less unchanged from 0.5 to 48 h of incubation. It is hard to derive a definitive conclusion from these observations as it is likely that due to binding of adiponectin with bFGF in the supernatants of cell culture interfered with the accurate detection. Another mitogenic growth factor EGF and over expression of its receptors has led to intensive research in developing drugs for blocking this carcinogenic activity [23]. However, in MDA-MB-436 and MFM-223 cells, we detected no expression of EGF either in the presence or absence of adiponectin. As discussed earlier, the vascularization in breast cancer is correlated directly with metastasis and cell invasion. Our data also showed that adiponectin-induced in vitro motility of breast cancer cells was substantially blocked by the inhibitors of MAPK and PI3 K. Clearly, these findings suggest that adiponectin promotes metastasis and invasion of cancer cells through MAPK and PI3 K signaling pathways. In summary, our results indicate that of adiponectin, dose dependently increased the in vitro motility of breast cancer cells, and it also augmented the expression of mitogenic and angiogenic growth factors, VEGF and TGFb1 in these cells.Certainly, these results do not explain as to why the obesity characterized by low circulating levels of adiponectin is a higher risk for cancer. Also, contrary to many reports, the mitogenic effects of adiponectin presently obtained suggest that further studies are needed to understand the role of adiponectin in linking obesity to breast cancer. The protective role of adiponectin against breast cancer was hypothesized and high levels of circulating adiponectin were associated with decreased risk of breast cancer first by Miyoshi et al. [24]. While this finding was supported by several of the subsequent retrospective case–control studies [7, 25, 26], many other investigations reported contradictory results suggesting that adiponectin was not strongly related to the risk of breast cancer [27]. Then, it was also
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indicated that blood levels of adiponectin were not associated with breast cancer independent of other known risk factors [28]. The inconsistency in the levels of female sex hormones, menstrual status etc. have been ascribed to the lack of a well defined association between adiponectin and breast cancer [29]. Several reports indicated that low levels of adiponectin are a high risk factor for breast cancer in postmenopausal but not in the premenopausal women [25– 27, 30]. A study based on meta-analysis including results of many investigations, indicated of inconsistency and therefore a difficulty in ascertaining if the effect of adiponectin on cancer promotion is negative or positive [31]. Our results further support that circulating adiponectin levels may not always be used as negative risk factor for breast cancer and related complications. It has also been shown that using a different antibody for immunoblotting, very little reduction in adiponectin expression related to stimulated adipogenesis was observed [22]. This finding raises the question as to whether the techniques used for the detection of adiponectin concentrations provide with an accurate estimation. Further studies are needed to define the role of adiponectin in promoting or suppressing the cancer-related signaling and its relation to hormone status, gender, age, and weight of the patient.
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