Norepinephrine upregulates VEGF, IL-8, and IL-6 expression in human melanoma tumor cell lines: implications for stress-related enhancement of tumor progression

Abstract

Studies suggest that stress can be a co-factor for the initiation and progression of cancer. The catecholamine stress hormone, norepinephrine (NE), may influence tumor progression by modulating the expression of factors implicated in angiogenesis and metastasis. The goal of this study was to examine the influence of NE on the expression of VEGF, IL-8, and IL-6 by the human melanoma cell lines, C8161, 1174MEL, and Me18105. Cells were treated with NE and levels of VEGF, IL-8, and IL-6 were measured using ELISA and real-time PCR. The expression of beta-adrenergic receptors (beta-ARs) mRNA and protein were also assessed. Finally, immunohistochemistry was utilized to examine the presence of beta1- and beta2-AR in primary and metastatic human melanoma biopsies. We show that NE treatment upregulated production of VEGF, IL-8, and IL-6 in C8161 cells and to a lesser extent 1174MEL and Me18105 cells. The upregulation was associated with induced gene expression. The effect on C8161 cells was mediated by both beta1- and beta2-ARs. Furthermore, 18 of 20 melanoma biopsies examined expressed beta2-AR while 14 of 20 melanoma biopsies expressed beta1-AR. Our data support the hypothesis that NE can stimulate the aggressive potential of melanoma tumor cells, in part, by inducing the production VEGF, IL-8, and IL-6. This line of research further suggests that interventions targeting components of the activated sympathetic-adrenal medullary (SAM) axis, or the utilization of beta-AR blocking agents, may represent new strategies for slowing down the progression of malignant disease and improving cancer patients' quality of life.

Figure 1

NE differentially modulates the levels of VEGF, IL-8, and IL-6 protein in culture supernatants of C8161 (A, B, and C), 1174MEL (D and E), and Me18105 (F and G) cells after treatment with NE. Levels of protein in culture supernatants were measured after treatment with 0, 0.1, 1, and 10 μM NE for 1, 3, 6, and 24 hours. Values are presented as percent of untreated control levels. ND, not detectable. Error bars represent the mean ± s.e.m. *, P ≤ 0.05; **, P ≤ 0.001.

Figure 2

Representative [3H]-thymidine incorporation assessing the effect of NE on cell proliferation. Cell proliferation was measured using [3H]-thymidine incorporation (see Materials and Methods) after C8161, 1174MEL, and Me18105 cells were treated with 0, 0.1, 1, and 10 μM NE for 24 hours. Six hours prior to the end of the 24 hour treatment, 10 μl of [³H]-thymidine (2 Ci/mmol) was added to medium. The labeling reaction was terminated and quantified using a liquid scintillation counter. Data are each expressed as mean ± s.e.m.; *, P ≤ 0.05; **, P ≤ 0.001.

Figure 3

The NE-dependent upregulation of VEGF, IL-8, and IL-6 mRNA levels in C8161 cells is mainly through the stimulation of transcription and not through increased mRNA stability. Representative real-time PCR assay measuring VEGF (A), IL-8 (B), and IL-6 (C) mRNA levels in C8161 cells after treatment with NE. Levels of mRNA in C8161 cells were measured using real-time PCR after treatment with 0, 0.1, 1, and 10 μM NE for 1, 2, 3, and 4 hours. Peak expression of VEGF mRNA was observed at 2 hours while highest levels of IL-8 and IL-6 mRNA levels were seen after 1 hour of treatment. Representative real-time PCR assay measuring VEGF (D), IL-8 (E), and IL-6 (F) mRNA levels in C8161 cells after treatment with 5 μg/ml actinomycin D (Act D), 10 μM NE (NE), or 10 μM NE plus 5 μg/ml actinomycin D (NE + Act D) for 2 hours (A) or 1 hour (B and C). Values are presented as percent of untreated control levels. Error bars represent the mean ± s.e.m; *, P ≤ 0.05; **, P ≤ 0.001.

Figure 4

Expression of β1-AR and β2-AR in C8161, 1174MEL and Me18105 melanoma cells. Representative real-time PCR assay measuring ADRB1 (A) and ADRB2 (B) mRNA levels in C8161, 1174MEL and Me18105 cells. Western blot analysis of β1-AR and β2-AR protein expression in C8161, 1174MEL and Me18105 melanoma cells. (C) Lysates from C8161, 1174MEL and Me18105 cells probed for β1-AR revealed a band with an apparent molecular weight of 75 × 10³ M_r ( ); 1174MEL and Me18105 cell lysates exhibit an additional band at 85 × 10³ M_r (←). (D) Several bands were observed when cell lysates were probed for β2-AR. A single band with molecular weight of about 47 × 10³ M_r was expressed in the three cell lines ( ) that is consistent with the unglycosylated protein. The band at about 65 × 10³ M_r (*) is the glycosylated receptor, while the bands at about 90 to100 × 10³ M_r are dimers (H). These bands were not observed in blots incubated with normal rabbit serum (not shown).

Figure 5

Assessment of the role of β-adrenergic receptors and the cAMP-PKA signaling pathway. Effect of β-blocker (propranolol) and a-blocker (phentolamine) on the NE-dependent modulation of VEGF, IL-8, and IL-6 mRNA in C8161, 1174MEL, and Me18105 cells. Representative real-time PCR assay measuring VEGF (A), IL-8 (B), and IL-6 (C) mRNA levels in C8161, 1174MEL, and Me18105 cells after treatment with 10 μM NE (NE), 10 μM phentolamine plus 10 μM NE (NE + Phen), or 1 μM propranolol plus 10 μM NE (NE + Prop) for 2 hours (A) or 1 hour (B and C) when peak gene expression were observed (See Fig. 3A–C). Representative real-time PCR assay measuring VEGF (D), IL-8 (E), and IL-6 (F) mRNA levels in C8161 cells after treatment with 10 μM NE (NE), 10 μM isoproterenol (Iso), 10 μM dobutamine (Dob), 10 μM terbutaline (Ter), 10 μM forskolin (Fsk), 100 μM 6-Bnz-cAMP (6-Bnz-cAMP), 100 μM 8-CPT (8-CPT), 10 μM H-89 plus 10 μM NE (NE + H89), or 10 μM myristoylated PKI plus 10 μM NE (NE + PKI) for for 2 hours (A) or 1 hour (B and C). Values are presented as percent of untreated control levels. Error bars represent the mean ± s.e.m; *, P ≤ 0.05; **, P ≤ 0.001.

Figure 6

A case of primary superficial-spreading melanoma illustrates intense β2-AR cytoplasmic/membrane immunoreactivity within the balls of melanoma cells (arrows) occupying the dermal/epidermal junction. The overlying epidermis and underlying dermis are non-immunoreactive. This staining was not apparent in negative control slides wherein no primary or secondary antibodies were applied (not shown). Bar = 200 μm.