In vitro impact of pegvisomant on growth hormone-secreting pituitary adenoma cells

Abstract

Pegvisomant (PEG), an antagonist of growth hormone (GH)-receptor (GHR), normalizes insulin-like growth factor 1 (IGF1) oversecretion in most acromegalic patients unresponsive to somatostatin analogs (SSAs) and/or uncontrolled by transsphenoidal surgery. The residual GH-secreting tumor is therefore exposed to the action of circulating PEG. However, the biological effect of PEG at the pituitary level remains unknown. To assess the impact of PEG in vitro on the hormonal secretion (GH and prolactin (PRL)), proliferation and cellular viability of eight human GH-secreting tumors in primary cultures and of the rat somatolactotroph cell line GH4C1. We found that the mRNA expression levels of GHR were characterized in 31 human GH-secreting adenomas (0.086 copy/copy β-Gus) and the GHR was identified by immunocytochemistry staining. In 5/8 adenomas, a dose-dependent inhibition of GH secretion was observed under PEG with a maximum of 38.2±17% at 1μg/mL (P<0.0001 vs control). A dose-dependent inhibition of PRL secretion occurred in three mixed GH/PRL adenomas under PEG with a maximum of 52.8±11.5% at 10μg/mL (P<0.0001 vs control). No impact on proliferation of either human primary tumors or GH4C1 cell line was observed. We conclude that PEG inhibits the secretion of GH and PRL in primary cultures of human GH(/PRL)-secreting pituitary adenomas without effect on cell viability or cell proliferation.

Keywords: GH4C1; acromegaly; pegvisomant; pituitary adenoma; prolactin.

Figure 1

Expression of the GHR in human GH-secreting adenomas. (A) mRNA expression of GHR assessed by real-time PCR in human liver (n=3), normal pituitary tissue (n=2), and GH-secreting pituitary adenomas (n=31). Note that 18 out of 31 adenomas display a higher mRNA level of GHR than the normal pituitary tissue. (B and C) GHR immunostaining as described in the ‘Design and methods’ section in a representative primary culture of a GH-secreting adenoma (63×, zoom factor 1) at T0 (B) and at T24 (hours) of pegvisomant 10μg/mL (C). Nucleus is stained in blue (DAPI) and GHR is shown as green spots (arrows). (D) Mean quantification of fluorescence of the GHR in both the cytoplasm and the nuclear compartment of GH-secreting cells from the tumor case no. 8. C/N, cytoplasm/nuclear ratio.

Figure 2

Cell viability of human GH-secreting adenomas (n=6) in primary culture. Cell viability was assessed by CellTiter-Glo (see ‘Design and methods’ section) under increasing doses of PEG ranging from 0.1 to 10μg/mL and measured after 72h of treatment. CTRL, control. Because of an insufficient amount of cells collected after dissection, tumors of cases 1 and 5 have not been included in this experiment.

Figure 3

Effect of PEG on hormonal secretions of primary cultures of human pituitary adenomas. (A and B) Mean dose–response GH suppression obtained in cell cultures from eight GH-secreting adenomas (Table 1) under PEG (0.1–10 μg/mL) categorized as five PEG-responsive tumors (A) (****P < 0.0001 vs CTRL) and three PEG-non-responsive tumors (B). (C) Correlation between maximal inhibition of GH secretion under PEG 1 μg/mL and GHR mRNA expression levels in eight GH-secreting adenomas (r = 0.68, P = 0.07). (D) Inhibition of GH secretion in two secreting adenomas (cases 1 and 4) after 72 h of octreotide 10^–9 mol/L alone (OCT 10^–9), PEG alone 10 μg/mL (PEG), or combination of both (OCT 10^–9π PEG). **P < 0.01. (E) Mean dose–response of PRL suppression obtained in cell cultures from three mixed GH/PRL adenomas (cases 1, 5, and 7) under PEG (0.1–10 μg/mL) ***P < 0.001, ****P < 0.0001 vs CTRL. (F) Western blot analysis of phospho-STAT5/STAT5 and phospho-JAK2/JAK2 in two GH-secreting adenomas (cases 2 and 8) after 5 min of treatment by PEG (10 μg/mL). Upper panel: representative blot of one tumor (case 2). Lower panel: quantification of both phospho-STAT5/STAT5 and phospho-JAK2/JAK2 on the immunoblot of the two tumors. *P < 0.05 vs CTRL.

Figure 4

Expression of rat GHR and cell viability and proliferation under pegvisomant in GH4C1 cell line. (A) Confocal microscopy (63×, zoom factor 1). Left panel, control without primary antibody. Right panel, rGHR is labeled in green (arrows) when cells were incubated with the primary (directed against rGHR) and the secondary antibody. Nucleus is stained in blue (DAPI). (B) No effect of 72h PEG (0.1–10μg/mL) on the cell viability of GH4C1 assessed by CellTiter-Glo (n=3 experiments). (C) Proliferation of GH4C1 cell lines assessed by EdU incorporation from day 1 to day 3 under PEG 10μg/mL compared with control (CTRL) NS, no significant. (D) Impact of PEG (10μg/mL) on the EdU incorporation/cell number ratio of GH4C1 cell lines after 3days, compared with control (CTRL).