Additionally, 8-CPT and forskolin (cAMP analogs) both raised VEGF, IL-8, and IL-6 mRNA levels implicating cAMP as a mediator. Lastly, H-89 (PKA inhibitor) nearly checked the effect of NE which could be just partially inhibited by PKI. To further identify the role of β-AR/cAMP/PKA signaling pathway in NE-treated A549 cells, the changes in VEGF, IL-8, and IL-6 protein levels tested by the ELISA assay related to mRNA levels as above were also analyzed. We observed similar changes in VEGF, IL-8, and

IL-6 protein levels with their selleck compound mRNA levels (Figure  5A-D). Figure 5 Evaluation of β-AR/cAMP/PKA signaling pathway by ELISA. The NE-dependent stimulation of VEGF, IL-8, and IL-6 protein levels could not be blocked by phentolamine (PHEN) (A). Representative results of VEGF (B), IL-8 (C), and IL-6 (D) protein levels treated with NE, isoproterenol (ISO), dobutamine (DOB), terbutaline (TER), 8-CPT, forskolin (FOR), NE + H89 or NE + PKI for 6 hours. Values are presented as percent of untreated control levels. Each BYL719 molecular weight bar represents the mean ± SD. *, P ≤ 0.05; **, P ≤ 0.001. We also evaluated the proliferation and migration of A549 cells under

the inhibitors PKI and H-89. The results showed that, different from PKI, H-89 inhibited the proliferation (Figure  6A) and migration (Figure  6B-C) of A549 cells. These results were consistent with the protein and gene levels of VEGF, IL-8 and IL-6 of A549 cells under PKI and H-89. Figure 6 The proliferation and migration of A549 cells under

PKI Progesterone and H-89. MTT assay showed that H-89 inhibited the proliferation of A549 cells (A) and wound healing assay showed H-89 lowered the migration of A549 cells (B and C). CON, control. *, P ≤ 0.05. Discussion In this study we showed that NE spurred tumor growth in the murine melanoma model treated with sunitinib by gavage in vivo and could be inhibited by propranolol. We also identified that NE upregulated VEGF, IL-8, and IL-6 protein levels in B16F1 cells in the presence or absence of the treatment with sunitinib at the concentration equal to IC50, which was blocked by propranolol. In addition, NE-dependent up-regulation in both protein and gene levels of VEGF, IL-8, and IL-6 was observed in human lung adenocarcinoma cells in which β-AR/cAMP/PKA signaling pathway was proved as the important mechanism. Chronic stress has been acknowledged as an important factor affecting patients with cancer and the effect of chronic stress may be persistent during the process from diagnosis for cancer to death of cancer. The activation on sympathetic nervous system by stress gives rise to the increased level of catecholamines resulting in several biological effects via ARs such as VEGF-caused stimulation in angiogenesis, raised levels of cytokines including IL-8 and IL-6 [42]. These effects were also proved in our study and found as at least a part of factors attenuating the efficacy of sunitinib in preclinical models.