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Recent studies have demonstrated that chronic stress promotes tumor growth, angiogenesis, and metastasis. In ovarian cancer, levels of the pro-angiogenic cytokine, interleukin 6 (IL-6), are known to be elevated in individuals experiencing chronic stress, but the mechanism(s) by which this cytokine is regulated and its role in tumor growth remain under investigation. Here we show that stress hormones such as norepinephrine lead to increased expression of IL-6 mRNA and protein levels in ovarian carcinoma cells. Furthermore, we demonstrate that norepinephrine stimulation activates Src tyrosine kinase and this activation is required for increased IL-6 expression. These results demonstrate that stress hormones activate signaling pathways known to be critical in ovarian tumor progression. Recent studies have demonstrated that chronic stress promotes tumor growth, angiogenesis, and metastasis. In ovarian cancer, levels of the pro-angiogenic cytokine, interleukin 6 (IL-6), are known to be elevated in individuals experiencing chronic stress, but the mechanism(s) by which this cytokine is regulated and its role in tumor growth remain under investigation. Here we show that stress hormones such as norepinephrine lead to increased expression of IL-6 mRNA and protein levels in ovarian carcinoma cells. Furthermore, we demonstrate that norepinephrine stimulation activates Src tyrosine kinase and this activation is required for increased IL-6 expression. These results demonstrate that stress hormones activate signaling pathways known to be critical in ovarian tumor progression. Chronic stress, which results in increased production of catecholamines such as norepinephrine (NE) 3The abbreviations used are: NE, norepinephrine; NK, natural killer; IL, interleukin; DMEM, Dulbecco's modified Eagle's medium; ELISA, enzyme-linked immunosorbent assay; RIPA, radioimmune precipitation buffer; PBS, phosphate-buffered saline; siRNA, short interfering RNA; DOPC, 1,2-dioleoyl-sn-glycero-3-phosphatidylcholine; Bt2cAMP, dibutyryl cyclic AMP. and epinephrine from the adrenal medulla and sympathetic neurons, has long been believed to adversely influence health (1Kemp V.H. Hatmaker D.D. Res. Nurs. Health. 1989; 12: 331-336Crossref PubMed Scopus (43) Google Scholar). While elevated levels of such stress mediators are known to be associated with consequences such as increased risk of heart disease and infection (2McEwen B.S. Neurobiol. Aging. 2002; 23: 921-939Crossref PubMed Scopus (556) Google Scholar, 3Sapolsky R.M. Why Zebras Don't Get Ulcers: A Guide to Stress, Stress-related Diseases, and Coping. W. H. Freeman and Co., New York1998Google Scholar, 4Weiner H. Perturbing the Organism: The Biology of Stressful Experience. University of Chicago Press, Chicago1992Google Scholar), several studies have indicated that chronic stress can also promote tumor development and progression (5Antoni M.H. Lutgendorf S.K. Cole S.W. Dhabhar F.S. Sephton S.E. McDonald P.G. Stefanek M. Sood A.K. Nat. Rev. Cancer. 2006; 6: 240-248Crossref PubMed Scopus (755) Google Scholar). For example, chronic stress has been associated with an increased incidence of mammary tumors in female mice carrying the Bittner oncogenic virus (6Riley V. Science. 1975; 189: 465-467Crossref PubMed Scopus (161) Google Scholar), and experimentally stressed mice exhibited accelerated development of ultraviolet-induced cutaneous tumors compared with non-stressed animals (7Parker J. Klein S.L. McClintock M.K. Morison W.L. Ye X. Conti C.J. Peterson N. Nousari C.H. Tausk F.A. J. Am. Acad. Dermatol. 2004; 51: 919-922Abstract Full Text Full Text PDF PubMed Scopus (33) Google Scholar, 8Saul A.N. Oberyszyn T.M. Daugherty C. Kusewitt D. Jones S. Jewell S. Malarkey W.B. Lehman A. Lemeshow S. Dhabhar F.S. J. Natl. Cancer Inst. 2005; 97: 1760-1767Crossref PubMed Scopus (151) Google Scholar). We have recently demonstrated that chronic stress resulted in greater tumor burden and markedly increased vascularization in orthotopic models of ovarian cancer (9Thaker P.H. Han L.Y. Kamat A.A. Arevalo J.M. Takahashi R. Lu C. Jennings N.B. Armaiz-Pena G. Bankson J.A. Ravoori M. Merritt W.M. Lin Y.G. Mangala L.S. Kim T.J. Coleman R.L. Landen C.N. Li Y. Felix E. Sanguino A.M. Newman R.A. Lloyd M. Gershenson D.M. Kundra V. Lopez-Berestein G. Lutgendorf S.K. Cole S.W. Sood A.K. Nat. Med. 2006; 12: 939-944Crossref PubMed Scopus (932) Google Scholar). These studies demonstrate that chronic stress influences tumor progression, but to date, the specific underlying mechanisms responsible for this outcome are poorly understood. A growing number of studies have demonstrated that chronic stress and other behavioral factors have specific effects on the immune system of cancer patients (10Glaser R. Kiecolt-Glaser J.K. Nat. Rev. Immunol. 2005; 5: 243-251Crossref PubMed Scopus (1488) Google Scholar). These effects include decreased natural killer (NK) cell cytotoxicity, weakened response of NK cells to recombinant interferon γ, and reduced T-cell response to mitogen stimulation (10Glaser R. Kiecolt-Glaser J.K. Nat. Rev. Immunol. 2005; 5: 243-251Crossref PubMed Scopus (1488) Google Scholar, 11Lutgendorf S.K. Sood A.K. Anderson B. McGinn S. Maiseri H. Dao M. Sorosky J.I. De Geest K. Ritchie J. Lubaroff D.M. J. Clin. Oncol. 2005; 23: 7105-7113Crossref PubMed Scopus (208) Google Scholar). However, additional factors are known to play a major role in tumor growth; therefore, we asked whether stress mediators could directly alter tumor cell production of cytokines known to contribute to tumor progression. Angiogenesis, the formation of new blood vessels from the preexisting vasculature, is necessary for tumor expansion and ascites formation and is regulated by the equilibrium of proangiogenic and antiangiogenic molecules (12Fidler I.J. J. Natl. Cancer Inst. 2001; 93: 1040-1041Crossref PubMed Scopus (93) Google Scholar, 13Folkman J. Semin. Oncol. 2002; 29: 15-18Crossref PubMed Scopus (2456) Google Scholar). Ovarian cancer cells have been shown to express β-adrenergic receptors, and stimulation of these cells with catecholamines results in enhanced expression of an important pro-angiogenic factor, vascular endothelial growth factor (9Thaker P.H. Han L.Y. Kamat A.A. Arevalo J.M. Takahashi R. Lu C. Jennings N.B. Armaiz-Pena G. Bankson J.A. Ravoori M. Merritt W.M. Lin Y.G. Mangala L.S. Kim T.J. Coleman R.L. Landen C.N. Li Y. Felix E. Sanguino A.M. Newman R.A. Lloyd M. Gershenson D.M. Kundra V. Lopez-Berestein G. Lutgendorf S.K. Cole S.W. Sood A.K. Nat. Med. 2006; 12: 939-944Crossref PubMed Scopus (932) Google Scholar, 14Lutgendorf S.K. Cole S. Costanzo E. Bradley S. Coffin J. Jabbari S. Rainwater K. Ritchie J.M. Yang M. Sood A.K. Clin. Cancer Res. 2003; 9: 4514-4521PubMed Google Scholar). These findings suggest that stress hormones may promote tumor angiogenesis. Interleukin-6 (IL-6) has been implicated as an important molecule in tumor progression and angiogenesis (15Nilsson M.B. Langley R.R. Fidler I.J. Cancer Res. 2005; 65: 10794-10800Crossref PubMed Scopus (276) Google Scholar). Elevated levels of IL-6 are frequently detected in the serum of ovarian cancer patients and are associated with a poor prognosis and increased tumor burden (16Plante M. Rubin S.C. Wong G.Y. Federici M.G. Finstad C.L. Gastl G.A. 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Cancer Res. 2001; 61: 8851-8858PubMed Google Scholar). The receptor for IL-6 (IL-6R) is expressed on endothelial cells, and stimulation of ovary and mesentery-derived endothelial cells with exogenous IL-6 results in enhanced cell migration in vitro (15Nilsson M.B. Langley R.R. Fidler I.J. Cancer Res. 2005; 65: 10794-10800Crossref PubMed Scopus (276) Google Scholar). Furthermore, IL-6 is a potent angiogenic cytokine in vivo (15Nilsson M.B. Langley R.R. Fidler I.J. Cancer Res. 2005; 65: 10794-10800Crossref PubMed Scopus (276) Google Scholar). Previously, IL-6 has been linked to chronic stress, in that individuals experiencing chronic stress have been shown to exhibit elevated circulating levels of IL-6 (22Kiecolt-Glaser J.K. Preacher K.J. MacCallum R.C. Atkinson C. Malarkey W.B. Glaser R. Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 9090-9095Crossref PubMed Scopus (935) Google Scholar). Moreover, behavioral factors have been associated with circulating and ascites IL-6 levels in ovarian cancer patients (23Costanzo E.S. Lutgendorf S.K. Sood A.K. Anderson B. Sorosky J. Lubaroff D.M. Cancer. 2005; 104: 305-313Crossref PubMed Scopus (167) Google Scholar). Epinephrine has been shown to increase IL-6 gene expression in adipose tissue (24Keller P. Keller C. Robinson L.E. Pedersen B.K. J. Appl. Physiol. 2004; 97: 1309-1312Crossref PubMed Scopus (22) Google Scholar), and norepinephrine infusion increases IL-6 expression in myocytes (25Briest W. Rassler B. Deten A. Leicht M. Morwinski R. Neichel D. Wallukat G. Ziegelhoffer T. Zimmer H.G. Pflugers Arch. Eur. J. Physiol. 2003; 446: 437-446Crossref PubMed Scopus (30) Google Scholar). However, whether tumor cell expression of IL-6 is influenced by chronic stress is unknown. In the current study, we evaluated the effect of stress-related hormones on IL-6 production by ovarian cancer cells and found that catecholamines enhance IL-6 promoter activity and protein levels. Moreover, the effect of norepinephrine on IL-6 expression is mediated through β-adrenergic receptors and requires the subsequent activation of the Src proto-oncogene product. Cell Culture—SKOV3.ip1 is a variant derived from SKOV3 cells (ATCC, Manassas, VA) that was established from the ascites fluid of female nude mice following intraperitoneal injection of the parental line (26Yu D. Wolf J.K. Scanlon M. Price J.E. Hung M.C. Cancer Res. 1993; 53: 891-898PubMed Google Scholar). The human ovarian cancer cell line Hey-A8 was a kind gift from Dr. Gordon B. Mills (The University of Texas M. D. Anderson Cancer Center, Houston, TX). EG cells have been described previously (14Lutgendorf S.K. Cole S. Costanzo E. Bradley S. Coffin J. Jabbari S. Rainwater K. Ritchie J.M. Yang M. Sood A.K. Clin. Cancer Res. 2003; 9: 4514-4521PubMed Google Scholar). Cell lines were grown as monolayers in Dulbecco's modified Eagle's medium (DMEM) containing 10% fetal bovine serum, vitamins, sodium pyruvate, l-glutamine, and nonessential amino acids (Invitrogen). To evaluate the effect of stress hormones on IL-6 secretion, 1.0 × 105 SKOV3.ip1, Hey-A8, and EG cells were seeded into individual wells of a 24-well plate. Following a 24-h incubation, triplicate cultures (wells) were stimulated with 500 μl of media containing norepinephrine, epinephrine, or the synthetic β-adrenergic receptor agonist, isoproterenol (Sigma), at 0-, 0.1-, 1-, and 10-μm concentrations. After 1, 3, and 6 h, medium was collected, centrifuged, and tested for the presence of IL-6 by ELISA (R PubMed Scopus Google Scholar, A. J. C. J. C. L. J. 2001; Scopus Google Scholar). IL-6 mRNA by was and with to the 10% of the was a in a with of at of at and of of and at by of at used for human and IL-6 were with gene as the in was with modified radioimmune precipitation as previously Jr., C.N. Lu C. Han L.Y. E. J. Mangala L.S. Merritt W.M. Lin Y.G. C. R. Kamat A.A. Li Y. P. Gershenson D.M. Sood A.K. J. Natl. Cancer Inst. 2006; PubMed Scopus Google Scholar). were a protein of cell protein was to and to a were with and with Systems, Minneapolis, at was detected and with a IL-6 SKOV3.ip1, and cells were with of a production of from a of the for the human were in serum to the cells were with for by for were in phosphate-buffered and for activity and a To of the IL-6 promoter responsible for stress we expression by of the human IL-6 promoter in the or as previously described O. A. R. M. M. P. M. J. Full Text Full Text PDF PubMed Scopus Google Scholar). and activation of β-adrenergic receptors, and Hey-A8 cells were seeded into 24-well as described were with the receptor at a of Following a incubation, medium was and cells were stimulated with 10% fetal bovine serum containing 10 with or for cells were with the or 100 of an of that activates for medium was collected, centrifuged, and tested for the presence of IL-6 by of Src cells × were seeded into in 10% fetal bovine the cells were in in the presence or of the Src for were stimulated for or with containing 10 in the presence or of cells were in were in PBS, and cells were by in sodium sodium with and sodium were by at × for 500 of protein in μl of was with 6 μl of μl of a of protein in was to and was for an additional were by with and by in of protein was on an a Cell and and with Src In mice were from The mice were and under specific conditions in by the for of in with current and of the U. S. of U. S. of and and the of Health. The mice were used to were of tumor cells were from cultures by a to and was with fetal bovine The cells were in medium and in with greater as by were used for the To × cells were into the of tumor cell mice were with of intraperitoneal isoproterenol or isoproterenol with or for were in a of tumor cell mice were were and was as × IL-6 from and into a as previously described J. Kamat A.A. Landen C.N. Han L.Y. Lutgendorf S.K. Lin Y.G. Merritt W.M. Jennings N.B. A. Coleman R. Gershenson D. R. Cole S.W. Lopez-Berestein G. Sood A.K. Clin. Cancer Res. 2006; 12: PubMed Scopus Google Scholar, Jr., C.N. A. C. R. Lopez-Berestein G. Sood A.K. Cancer Res. 2005; 65: PubMed Scopus Google Scholar). in IL-6 levels were with an of factors as A was of Ovarian Cancer IL-6 the effect of stress hormones on IL-6 expression in ovarian tumor cell SKOV3.ip1, Hey-A8, and EG cells were stimulated with of norepinephrine, epinephrine, or and were for IL-6 by ELISA 1, In human ovarian carcinoma cell lines stimulated with isoproterenol or stress levels of norepinephrine or epinephrine, a increase in IL-6 stimulation of cells with norepinephrine resulted in a increase in IL-6 production at the The level of IL-6 by cells at was and with and 10 norepinephrine IL-6 levels increased to and norepinephrine to a increase in IL-6 by EG cells and a increase in IL-6 by Hey-A8 cells of human ovarian cancer cell lines with epinephrine also increased IL-6 Hey-A8 cells with 10 epinephrine of in a increase in to Epinephrine an increase in IL-6 in and EG cells Moreover, the synthetic β-adrenergic receptor isoproterenol also a increase in IL-6 Following of Hey-A8 and cells, we an increase in IL-6 expression of the cells with norepinephrine or epinephrine the increases in IL-6 protein with ELISA enhance IL-6 by tumor cells. ovarian cancer cell lines Hey-A8, and were stimulated with 0.1-, 1-, or 10-μm of norepinephrine epinephrine or isoproterenol was or 6 and tested for the presence of IL-6 by are as increase in IL-6 to level of IL-6 with cells were stimulated with norepinephrine 10 or isoproterenol 10 for h, and protein was from cell for an IL-6 The of to is shown on the IL-6 the mechanisms by which catecholamines IL-6 production by ovarian cancer cells, we in cells were stimulated with 10 norepinephrine, and IL-6 mRNA levels were at from to The level of IL-6 mRNA was levels. shown in norepinephrine increased IL-6 mRNA by at 6 h, and IL-6 mRNA levels to line a effect on other ovarian cancer cell lines IL-6 whether increased activity of the promoter was responsible for norepinephrine of gene we the effects of norepinephrine on a by of human of the O. M. R. V. J. Johnson M. J. Full Text Full Text PDF PubMed Scopus Google Scholar). this was into cells, norepinephrine enhanced production by that increased IL-6 mRNA was to increased increased effects were in SKOV3 and Hey-A8 cell lines stimulation of the signaling with also enhanced activity of the that the effects of norepinephrine be mediated by the receptors that activate the The by was in to that by the protein kinase which as a in these To the effects of norepinephrine at the IL-6 protein levels in cells were evaluated in the presence of effect on the of the IL-6 protein These results that norepinephrine increases IL-6 levels by activation by protein of IL-6 by of ovarian carcinoma cell lines express and receptors that can activate the signaling through stimulation of (14Lutgendorf S.K. Cole S. Costanzo E. Bradley S. Coffin J. Jabbari S. Rainwater K. Ritchie J.M. Yang M. Sood A.K. Clin. Cancer Res. 2003; 9: 4514-4521PubMed Google Scholar). To whether IL-6 expression could be regulated through β-adrenergic receptors, and Hey-A8 cells were with a β-adrenergic receptor for to stimulation with 10 norepinephrine or shown in of IL-6 by norepinephrine and isoproterenol was by These effects were specific to β-adrenergic receptors, as the effect on the of stress hormones to IL-6 production β-adrenergic receptors are known to we stimulated and Hey-A8 cells with the and found an increase in IL-6 production to that by norepinephrine of cells with resulted in a increase in IL-6 expression and an increase in Hey-A8 IL-6 production stimulation of the β-adrenergic receptors has been shown previously to also lead to activation of protein directly by M. P. D. V. A. E. J. Physiol. 2004; PubMed Scopus Google Scholar, M. M.B. D. A. K. F. O. S. M. J. 2004; Full Text Full Text PDF PubMed Scopus Google Scholar), we to whether activation of could contribute to IL-6 expression in human ovarian carcinoma cells. The and Hey-A8 cells were with 100 a that In cell increase of IL-6 of IL-6 by a role for the protein tyrosine kinase Src in β-adrenergic receptor W.L. J. Dermatol. 2004; Full Text Full Text PDF PubMed Scopus Google Scholar). on IL-6 by Src J.M. J.M. J. J. Full Text Full Text PDF PubMed Scopus Google Scholar, M. M.G. Am. J. Physiol. 2006; PubMed Scopus Google Scholar), we asked whether the IL-6 production was Following serum for h, the cells were with 10 norepinephrine in medium for that by 10 Src and the increase in Src at of increased Src the J.M. C.H. Y. R. J.A. Cancer 2005; PubMed Scopus Google Src To whether Src is required for the stimulation of IL-6 production by norepinephrine, cells were in medium or medium containing for and stimulated for with medium containing 10 norepinephrine in the presence or of ELISA of IL-6 in medium was The results demonstrated that of Src activity by the effect of norepinephrine on IL-6 Furthermore, IL-6 expression in cells. results were and of Src in of the whether activation of the promoter required Src SKOV3 or Hey-A8 cells were with or for to shown in of Src of promoter of promoter in factor that activation a However, of the of the effect was specific to as the promoter response to the These suggest that β-adrenergic activation of the promoter the Src signaling is mediated by response to kinase A signaling but activation of of IL-6 in in the role of IL-6 in tumor growth, we used a for in vivo that we have previously for in vivo J. Kamat A.A. Landen C.N. Han L.Y. Lutgendorf S.K. Lin Y.G. Merritt W.M. Jennings N.B. A. Coleman R. Gershenson D. R. Cole S.W. Lopez-Berestein G. Sood A.K. Clin. Cancer Res. 2006; 12: PubMed Scopus Google Scholar, Jr., C.N. A. C. R. Lopez-Berestein G. Sood A.K. Cancer Res. 2005; 65: PubMed Scopus Google Scholar). We an that decreased IL-6 levels in the cells female mice were with cells and to the following intraperitoneal IL-6 intraperitoneal isoproterenol intraperitoneal IL-6 isoproterenol intraperitoneal with increased tumor by IL-6 reduced tumor growth by in the and the tumor growth that chronic stress hormones expression of a pro-angiogenic cytokine in ovarian carcinoma cells, and through a β-adrenergic tyrosine kinase signaling For these we stimulated ovarian cancer cells with physiologic and found that for of the cell lines tested Hey-A8, and norepinephrine and epinephrine increased IL-6 Moreover, we have demonstrated that of cells with norepinephrine results in increases in IL-6 and promoter that stress mediators expression of the gene at the The effects of catecholamines are mediated through to β-adrenergic receptors on the of cells. results in activation of which the formation of the from to and activates protein kinase to the of molecules L. V. J.K. Am. J. Physiol. PubMed Google Scholar). In our studies the effect of catecholamines on IL-6 expression was by the of the β-adrenergic receptor but by the receptor by as a of as a of and as a specific of the we that IL-6 expression through an Recent have demonstrated that activation of β-adrenergic receptors results in enhanced Src activity M. O. M.G. M. V. J. 2002; Full Text Full Text PDF PubMed Scopus Google Scholar, J. S. W. Full Text Full Text PDF PubMed Scopus Google Scholar). we evaluated the role of Src in IL-6 expression and that norepinephrine stimulation to an increase in Src Moreover, the Src and the of IL-6 expression and activation of the human promoter by These that stress-related hormones activate β-adrenergic receptors on ovarian cancer cells by the kinase activity of which results in enhanced of the promoter and subsequent production of IL-6 human ovarian cancer by have demonstrated that Src is in the of ovarian Bast R.C. Mills Gynecol. Oncol. 2003; Full Text PDF PubMed Scopus Google and to angiogenesis in these the mechanism(s) by which Src is are The current a by which Src may be in these by response to stress hormones through β-adrenergic activation of Src has been shown to enhance expression of vascular endothelial growth factor J.M. J.M. J. J. Full Text Full Text PDF PubMed Scopus Google and J.M. M.B. C.H. Cancer Res. 2005; 65: PubMed Scopus Google Scholar), catecholamines may increase tumor cell production of these proangiogenic molecules through a that norepinephrine levels increase in that catecholamines play a role in the of the normal ovary M. M. S.L. A. A.A. Res. 2002; PubMed Scopus Google Scholar). cells may these mechanisms to promote disease progression. Epinephrine and norepinephrine have been demonstrated to IL-6 expression in adipose cells and myocytes (24Keller P. Keller C. Robinson L.E. Pedersen B.K. J. Appl. Physiol. 2004; 97: 1309-1312Crossref PubMed Scopus (22) Google Scholar, W. Rassler B. Deten A. Leicht M. Morwinski R. Neichel D. Wallukat G. Ziegelhoffer T. Zimmer H.G. Pflugers Arch. Eur. J. Physiol. 2003; 446: 437-446Crossref PubMed Scopus (30) Google Scholar). findings are with these and a by which stress hormones enhance tumor production of In other catecholamines have been shown to ovarian cancer cell expression of vascular endothelial growth factor (14Lutgendorf S.K. Cole S. Costanzo E. Bradley S. Coffin J. Jabbari S. Rainwater K. Ritchie J.M. Yang M. Sood A.K. Clin. Cancer Res. 2003; 9: 4514-4521PubMed Google Scholar). these that activation of β-adrenergic pathways on ovarian tumor cells can enhance expression of proangiogenic molecules critical to ovarian cancer progression
Nilsson et al. (Fri,) studied this question.