Growth hormone releasing hormone induces the expression of nitric oxide synthase

Abstract

Growth hormone releasing hormone (GHRH) and its receptors are expressed in a wide variety of human tumours and established cancer cell lines and are involved in carcinogenesis. In addition, GHRH antagonists exert an antitumour activity in experimental cancer models. Recent studies indicate that the mechanisms involved in the mediation of the effects of GHRH include the regulation of the metabolism of the reactive oxygen species. This work demonstrates the expression of GHRH receptors and GHRH in the A549 human lung cancer cell line and shows that the mitogenic effect of GHRH in these cells is dependent on the activation of the extracellular receptor kinase (ERK)1/2 pathway. The action of GHRH can be suppressed by GHRH antagonist MZ-5-156 and mitogen activated protein kinase (MAPK) inhibitor PD 098059. These results are reflected in the effect in the proliferating cell nuclear antigen. In addition, our study shows that GHRH increases the expression of the inducible nitric oxide synthase, an enzyme which is strongly involved in various human diseases, including cancer and augments key intracellular regulators of its expression, such as pNF (nuclear factor)κBp50 and cyclooxygenase 2. GHRH antagonist MZ-5-156 counteracts the effects of GHRH in these studies, indicating that this class of peptide antagonists may be useful for the treatment of diseases related to increased oxidative and nitrosative stress.

fig 1

(A) Western blot analysis of the expression of GHRH receptor(s) in A549 lung cancer, LNCaP prostate cancer cell line and 3T3 mouse fibroblast cell line. LNCaP and 3T3 cells were used as positive and negative controls, respectively. (B) Western blot analysis of the expression of GHRH receptor(s) in T47D breast cancer cells and 3T3 mouse fibroblast cell line. T47D cells were used as positive control. (C) Western blot analysis of the expression of GHRH in LNCaP, A549 and T47D cancer cell lines. LNCaP and T47D cells were used as positive controls.

fig 2

Western blot analysis of the pERK1/2 after incubation of the A549 cells with GHRH antagonist MZ-5–156 and GHRH. The protein levels were normalized to ERK2 signal (loading control). The blot is representative of two independent experiments.

fig 3

(A) Proliferation rate of the A549 cells exposed to 0.1 μM and 1 μM GHRH (1–29)NH2 and MZ-5–156 as well as 10 μM MAPK inhibitor. *P < 0.01 versus control cells **P < 0.001 versus control cells. NS: non-significant. (B) Proliferation rate of the 3T3 cells exposed to 0.1 μM and 1 μM GHRH (1–29)NH₂ and MZ-5–156 as well as 10 μM MAPK inhibitor. NS: non–significant.

fig 4

Expression of PCNA by A549 (top) or 3T3 (bottom) cells after exposure to 0.1 μM or 1 μM GHRH (1–29)NH₂ and 0.1 μM or 1 μM GHRH antagonist MZ-5–156. Protein levels were normalized to β actin signal (loading control). The blot is representative of two independent experiments.

fig 5

Expression of P53 by A549 cells after exposure to 0.1 μM or 1 μM GHRH (1–29)NH₂ and 0.1 μM GHRH antagonist MZ-5–156. Protein levels were normalized to β actin signal (loading control). The blot is representative of two independent experiments.

fig 6

(A) Expression of iNOS by A549 cells after exposure to 0.1 μM or 1 μM GHRH (1–29)NH₂ and 0.1 μM GHRH antagonist MZ-5–156. Protein levels were normalized to β actin signal (loading control). The blot is representative of two independent experiments (B) Expression of iNOS by 3T3 cells after exposure to 0.1 μM or 1 μM GHRH (1–29)NH₂. Protein levels were normalized to β actin signal (loading control). The blot is representative of two independent experiments,

fig 7

Effect of GHRH and MZ-5–156 on the expression of the COX-2 (upper panel) and the activation of the NF-κBp50 (lower panel) in A549 cells. The blot is representative of two independent experiments.