GHRH Drives iNOS, NF-κB, and COX-2 Expression in Lung Cancer Cells; Antagonist MZ-5-156 Blocks Effect
Background
Growth hormone releasing hormone (GHRH) and its receptors are aberrantly expressed across a wide range of human tumours and established cancer cell lines, implicating the peptide directly in carcinogenesis beyond its classical hypothalamic role. Prior work established that GHRH receptor signalling drives tumour cell proliferation and that GHRH antagonists can suppress experimental cancer growth, yet the downstream molecular mediators remained incompletely defined. Emerging evidence pointed toward reactive oxygen species (ROS) metabolism as one such mediator, but whether GHRH could directly engage the nitrosative stress axis — specifically inducible nitric oxide synthase (iNOS) and its transcriptional regulators — had not been demonstrated. Clarifying this link is critical because iNOS-driven nitrosative stress is broadly implicated in tumour progression, inflammation, and resistance to therapy.
Study Design
Experiments were conducted in the A549 human lung cancer cell line, which was first characterised for endogenous expression of GHRH and its receptors. The mitogenic response to GHRH was assessed using proliferating cell nuclear antigen (PCNA) as a readout of cell proliferation. The ERK1/2 pathway's role was interrogated with the MAPK inhibitor PD 098059, and receptor-specific effects were confirmed by co-treatment with GHRH antagonist MZ-5-156. Expression of iNOS, transcription factor pNF-κBp50, and COX-2 were evaluated as downstream endpoints of GHRH signalling, with the antagonist included as a suppression control across all endpoints.
Results
A549 cells were confirmed to express both GHRH and functional GHRH receptors, establishing an autocrine/paracrine signalling loop. GHRH exerted a mitogenic effect in these cells, and this proliferative response was dependent on activation of the ERK1/2 pathway, as demonstrated by its suppression with PD 098059. The increase in proliferation was reflected in elevated proliferating cell nuclear antigen (PCNA) levels. Beyond proliferation, GHRH significantly increased the expression of inducible nitric oxide synthase (iNOS), linking the peptide to the nitrosative stress axis for the first time in this model.
GHRH also augmented key intracellular regulators of
iNOSexpression — specifically pNF-κBp50 and COX-2 — indicating a coordinated transcriptional programme driving oxidative and nitrosative stress downstream of GHRH receptor activation.
GHRH antagonist MZ-5-156 counteracted all of these effects — the mitogenic response, ERK1/2-dependent proliferation, PCNA elevation, iNOS upregulation, and induction of pNF-κBp50 and COX-2 — demonstrating that these actions are specifically receptor-mediated and blockable by peptide antagonism.
Key Findings
- A549 human lung cancer cells express both GHRH and functional GHRH receptors, forming an autocrine loop.
- GHRH mitogenic effect in A549 cells is dependent on ERK1/2 pathway activation; blocked by PD 098059.
- GHRH increases iNOS expression, linking GHRH signalling to the nitrosative stress axis in lung cancer.
- GHRH upregulates pNF-κBp50 and COX-2, key transcriptional regulators of iNOS expression.
- GHRH antagonist MZ-5-156 suppresses all GHRH-driven effects: proliferation, iNOS, NF-κB, and COX-2.
Why It Matters
GHRH antagonists such as MZ-5-156 may have therapeutic utility not only as antiproliferatives but as suppressors of iNOS-driven nitrosative and oxidative stress in lung cancer, a dual mechanism that broadens their potential application beyond tumour growth inhibition. For clinicians and translational researchers, the identification of iNOS, NF-κB, and COX-2 as GHRH-regulated targets suggests that diseases characterised by elevated nitrosative stress — including inflammatory cancers — could be amenable to GHRH antagonist-based intervention. The ERK1/2/MAPK dependency provides a molecularly defined pathway that may allow patient stratification or combination strategies pairing GHRH antagonists with existing MAPK inhibitors. The finding that an autocrine GHRH loop operates in A549 cells reinforces the case for targeting local tumour GHRH signalling, independent of hypothalamic GHRH physiology, which is the context most relevant to sermorelin-class peptides used clinically.
