Abstract
A growing number of studies indicate that chronic stress can accelerate tumor growth due to sustained sympathetic nervous system activation. Our recent findings suggest that chronic stress is associated with increased IL8 levels. Here, we examined the molecular and biological significance of IL8 in stress-induced tumor growth. Norepinephrine (NE) treatment of ovarian cancer cells resulted in a 250-300% increase in IL8 protein and 240-320% increase in its mRNA levels. Epinephrine treatment resulted in similar increases. Moreover, NE treatment resulted in a 3.5-4-fold increase in IL8 promoter activity. These effects were blocked by propranolol. Promoter deletion analyses suggested that AP1 transcription factors might mediate catecholamine-stimulated up-regulation of IL8. siRNA inhibition studies identified FosB as the pivotal component responsible for IL8 regulation by NE. In vivo chronic stress resulted in increased tumor growth (by 221 and 235%; p < 0.01) in orthotopic xenograft models involving SKOV3ip1 and HeyA8 ovarian carcinoma cells. This enhanced tumor growth was completely blocked by IL8 or FosB gene silencing using 1,2-dioleoyl-sn-glycero-3-phosphatidylcholine nanoliposomes. IL8 and FosB silencing reduced microvessel density (based on CD31 staining) by 2.5- and 3.5-fold, respectively (p < 0.001). Our findings indicate that neurobehavioral stress leads to FosB-driven increases in IL8, which is associated with increased tumor growth and metastases. These findings may have implications for ovarian cancer management.
Highlights
Obstetrics and Gynecology, Samsung Medical Center, Sungkyunkwan University School of Medicine, Seoul 135-710, Korea, and the §§Division of Hematology-Oncology, Department of Medicine, UCLA, Los Angeles, California 90095
In vivo chronic stress resulted in increased tumor growth in orthotopic xenograft models involving SKOV3ip1 and HeyA8 ovarian carcinoma cells
Our findings indicate that neurobehavioral stress leads to FosB-driven increases in IL8, which is associated with increased tumor growth and metastases
Summary
Cell Lines and Culture Conditions—The HeyA8 and SKOV3ip ovarian cancer cell lines were propagated as described previously [25]. Enzyme-linked Immunosorbent Assay (ELISA)—IL8 protein levels in the supernatant collected from cells treated with either NE or Epi were quantified by ELISA, using the Quantikine Immunoassay kit (R & D Systems, Minneapolis, MN) according to the manufacturer’s protocol and as described previously [24]. IL8 Promoter Analysis—HeyA8 or SKOV3ip (3 ϫ 105) cells were transfected (Lipofectamine 2000; Invitrogen) with a fulllength sequence [26, 27] of IL8 promoter (1481 bp upstream of transcription start site) and subsequently assayed for luciferase reporter gene expression (Promega) after 3 h of exposure to vehicle control or pharmacological agonists/antagonists of -adrenergic receptors or PKA. IL8 promoter activity was assayed by expression of a luciferase reporter gene in HeyA8 ovarian cancer cells after 3 h of exposure to NE (10 M). To quantify microvessel density (MVD), we examined 10 random 0.159-mm fields at ϫ100 magnification for each tumor and counted the microvessels within those fields [25]
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