Role of endothelial nitric oxide synthase (eNOS) in chronic stress-promoted tumour growth

Abstract

Accumulating evidence suggests that chronic stress can be a cofactor for the initiation and progression of cancer. Here we evaluated the role of endothelial nitric oxide synthase (eNOS) in stress-promoted tumour growth of murine B16F10 melanoma cell line in C57BL/6 mice. Animals subjected to restraint stress showed increased levels adrenocorticotropic hormone, enlarged adrenal glands, reduced thymus weight and a 3.61-fold increase in tumour growth in respect to no-stressed animals. Tumour growth was significantly reduced in mice treated with the β-antagonist propranolol. Tumour samples obtained from stressed mice displayed high levels of vascular endothelial growth factor (VEGF) protein in immunohistochemistry. Because VEGF can induce eNOS increase, and nitric oxide is a relevant factor in angiogenesis, we assessed the levels of eNOS protein by Western blot analysis. We found a significant increase in eNOS levels in tumour samples from stressed mice, indicating an involvement of this enzyme in stress-induced tumour growth. Accordingly, chronic stress did not promote tumour growth in eNOS(-/-) mice. These results disclose for the first time a pivotal role for eNOS in chronic stress-induced initiation and promotion of tumour growth.

Fig 1

Measurements of ACTH levels and weight of thymus in stressed mice. Measurements of ACTH levels on peripheral blood sera obtained from control and stressed animals (n= 10) revealed that the levels of ACTH in stressed+placebo mice are increased respect to control mice P < 0.05 data are represented as the mean ± S.E. (n= 10). On the contrary, in stressed+placebo mice the weight of thymus is lower than the control mice and propranolol-treated mice. Data are represented as the mean ± S.E.; P < 0.05 (n= 10). Ppl: propranolol; ACTH: adrenocorticotropic hormone.

Fig 2

Effect of chronic stress on murine melanoma cancer growth. In vivo tumour growth of B16F10 cells was measured in C57BL/6 mice. The animals were distributed into three groups (10 animals/group). Two groups of animals received a pre-treatment with placebo (PBS) or with propranolol for seven days and then exposed to a physical restraint. After seven days, the animals were injected subcutaneously with 3 χ 10⁵ cells/mouse in the right hind footpad B16F10 cells. Differences in growth were analysed by ANOVA (n= 10; P < 0.0001). Mean tumour volume ± S.E.M. are given.

Fig 3

Three-dimensional measure and visualization of tumours. Coronal (A–C) post-contrast T1-weighted images (T1-w), applying intensity colouring maps of stressed + Ppl (A), stressed + placebo (B) and placebo (C). Dotted lines (A–C) and asterisks (D–F) indicate maximum enhancement plotted along X-axis on T1-w post-contrast images. MIP images show tumour lesions (arrows) and their extension throw a polygonal reconstruction of involved tissues (G–I). Volume images are representative of tumours of mice treated with stress + Ppl; stress + placebo and control. Volumes measured 0.349 cm³ (stressed + Ppl), 2.849 cm³ (stressed + placebo) and 0.1720 cm³ (control), respectively. Virtual renderings (L–N) cut on axial planes show a three-dimensional evaluation of tumours spatial extension and their relationships with normal tissues. Major axis is reported in cm in all images (G–I). Ppl: propranolol.

Fig 4

VEGFA expression in stressed mice. Melanoma sections from control and stressed mice were obtained and VEGFA levels were analysed by IHC. Protein lysated from the sections were obtained and VEGFA content was analysed by Western blotting. An anti–β-actin antibody was used as loading control (upper panel). Graphical representation of VEGFA/β-actin ratio in control and stressed mice (lower). Data are represented as the mean ± S.E.; (n= 3) *P= 0.01 versus ctrl and stress + Ppl; (n= 3) °P= 0.002 versus stress + placebo and stress + Ppl. Densitometry analysis was obtained from samples of two separate experiments. Scale bar: 100 μm in A–C. Ppl: propranolol.

Fig 5

eNOS expression in stressed mice. (A) Western blot analysis with anti-eNOS on tumour lysates from control and stressed mice revealed an increased level of eNOS in stressed + placebo mice compared to control; *°P= 0.02 and stressed + Ppl mice; *P= 0.002 (n= 4). β-Actin protein was analysed as loading control. (B) Graphical representation of eNOS/β-actin ratio in control and stressed mice. The results were obtained from two independent experiments. (C) No differences in tumour growth are observed in eNOS^−/− stressed and no stressed mice. (D) Tissue samples from B16F10 tumours implanted in eNOS^−/− mice either untreated or subjected to physical stress were processed and analyzed by Western blot for eNOS contents. This analysis revealed the presence of eNOS protein only in the positive control. Ppl: propranolol.