Imatinib Inhibits GH Secretion From Somatotropinomas

Abstract

Background: Imatinib, a tyrosine kinase inhibitor, causes growth failure in children with chronic myeloid leukemia probably by targeting the growth hormone (GH)/insulin like growth factor-1 (IGF-1) axis. We aim to explore the imatinib targets expression in pituitary adenomas and study the effect of imatinib on GH secretion in somatotropinoma cells and GH3 cell line. Materials and Methods: The expression pattern of imatinib's targets (c-kit, VEGF, and PDGFR-α/β) was studied using immunohistochemistry and immunoblotting 157 giant (≥4 cm) pituitary adenomas (121 non-functioning pituitary adenomas, 32 somatotropinomas, and four prolactinomas) and compared to normal pituitary (n = 4) obtained at autopsy. The effect imatinib on GH secretion, cell viability, immunohistochemistry, electron microscopy, and apoptosis was studied in primary culture of human somatotropinomas (n = 20) and in rat somato-mammotroph GH3 cell-line. A receptor tyrosine kinase array was applied to human samples to identify altered pathways. Results: Somatotropinomas showed significantly higher immunopositivity for c-kit and platelet-derived growth factor receptor-β (PDGFR-β; P < 0.009 and P < 0.001, respectively), while staining for platelet-derived growth factor receptor-α (PDGFR-α) and vascular endothelial growth factor (VEGF) revealed a weaker expression (P < 0.001) compared to normal pituitary. Imatinib inhibited GH secretion from both primary culture (P < 0.01) and GH3 cells (P < 0.001), while it did not affect cell viability and apoptosis. The receptor tyrosine kinase array showed that imatinib inhibits GH signaling via PDGFR-β pathway. Conclusion: Imatinib inhibits GH secretion in somatotropinoma cells without affecting cell viability and may be used as an adjunct therapy for treating GH secreting pituitary adenomas.

Keywords: PDGFR-α/β; VEGF; c-kit; growth hormone; imatinib; somatotropinoma.

Figure 1

(a) Bar diagram showing reduction in growth hormone levels in culture media after treatment with 0.5 μM imatinib in primary culture of somatotropinoma cells (P < 0.0001). (b,c) Representative image of immunohistochemistry with anti-GH antibody on treated and untreated primary somatotropinoma culture. (d) Bar diagram showing reduction in GH levels in culture media of GH3 cell line after treatment with 0.5 μM imatinib (P < 0.01). (e,f) IHC of GH3 cells showing intense GH positivity in untreated and no GH staining in treated cells (20X). (g,h) Ultra-structural analysis of GH granules using electron microscopy between treated and untreated GH3 cells (6000X). **P < 0.01; ***P < 0.0001.

Figure 2

Immunohistochemistry (IHC) of pituitary adenoma shows differential expression of tyrosine kinases (c-kit, VEGF, and PDGFR-α/β). Cytoplasmic immunopositivity for c-Kit was high in somatotropinoma followed by NFPA, prolactinoma, and normal pituitary (a–d). Strong cytoplasmic expression for VEGF was observed in NFPA as compared to somatotropinomas, prolactinomas and normal pituitaries (e–h). PDGFR-α expression was weakly positive in NFPA as compared to normal pituitary while it was negative for somatotropinoma and prolactinomas. (i–l), PDGFR-β expression was strongly positive in somatotropinomas as compared to normal pituitary while it was weakly positive in NFPA and prolactinomas (m–p).

Figure 3

Graph showing percentage of cytoplasmic TK (c-Kit, VEGF, PDGFR-α/β) immunostaining in normal pituitary (n = 4), NFPAs (n = 121), somatotropinomas (n = 32), and prolactinomas (n = 4). c-Kit showed moderate to strong positivity in somatotropinomas. VEGF showed higher percentage of positivity in NFPA. PDGFR-β was strongly positive in maximum number of somatotropinomas (59%), whereas PDGFR-α was negative (65%).

Figure 4

Double immunofluoroscence showing co-expression (arrows) of PDGFR-α and PDGFR-β (green) and of hGH (red) in normal pituitary gland (A,B,G,H) and in somatotropinoma (D,E,J,K). Overlays of the green and red channels are shown in the third column. (C′,F′,I′,L′) are the magnified views of the boxes marked in (C,F,I,L) respectively. Scale bar = 100 μm (A–L), 50 μm (C′,F′,I′,L′).

Figure 5

Quantification of tyrosine kinases (c-Kit, VEGF, PDGFR-α, and PDGFR-β) using western blot in (A) normal pituitary (n = 9), (B) NFPA (n = 27), and (C) somatotropinomas (n = 27). Data were normalized to β-Actin and compared to expression in normal pituitary. (D) Similar to IHC findings, PDGFR-β and c-Kit was overexpressed in somatotropinoma (P < 0.01) compared to normal pituitary whereas PDGFR-α was under expressed in somatotropinoma (P < 0.05). *P < 0.05; **P < 0.01 and ***P < 0.001.

Figure 6

Phospho-tyrosine profiling of somatotropinomas treated and untreated with 0.5 μM Imatinib. **P < 0.01. (A) Phospho-tyrosine profiling of somatotropinomas treated and untreated with 0.5 μM Imatinib. Note the absence of PDGFR-β after imatinib treatment (B) Bar diagram showing reduction in PDGFR-β levels after treatment with 0.5 μM imatinib in primary culture of somatotropinoma cells (P < 0.01).