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. 2022 Jan;45(1):29-41.
doi: 10.1007/s40618-021-01609-1. Epub 2021 Jun 14.

Long-term effects of somatostatin analogues in rat GH-secreting pituitary tumor cell lines

A Dicitore  1 , D Saronni  2 , G Gaudenzi  1 , S Carra  3 , M C Cantone  2 , M O Borghi  4   5 , L Persani  2   3 , G Vitale  6   7
Affiliations

Long-term effects of somatostatin analogues in rat GH-secreting pituitary tumor cell lines

A Dicitore et al. J Endocrinol Invest. 2022 Jan.

Abstract

Purpose: First-generation somatostatin analogs, octreotide (OCT) and lanreotide, are the cornerstone for the medical treatment of growth hormone (GH)-secreting pituitary tumors. A new multireceptor analog, such as pasireotide (PAS), showed better activity than OCT in long-term treatment of patients with acromegaly, but modulation of intracellular key processes is still unclear in vitro. In this study, we evaluated the antitumor activity of OCT and PAS in two GH-secreting pituitary tumor cell lines, GH3 and GH4C1, after a long-term incubation.

Methods: The effects of PAS and OCT on the cell viability, cell cycle, apoptosis, GH secretion, and tumor-induced angiogenesis have been evaluated through a colorimetric method (MTS Assay), DNA flow cytometry with propidium iodide, and Annexin V-FITC/propidium iodide staining, ELISA assay and zebrafish platform, respectively.

Results: PAS showed a more potent antitumor activity compared to OCT in GH3 cell line exerted through inhibition of cell viability, perturbation of cell cycle progression, and induction of apoptosis after 6 days of incubation. A concomitant decrease in GH secretion has been observed after 2 days of incubation only with PAS. No effect on tumor-induced angiogenesis has been reported after treatment with OCT or PAS in zebrafish/tumor xenograft model.

Conclusion: Long-term incubation with PAS showed a more potent antitumor activity than that reported after OCT in GH3 cells, mainly modulated by a cell cycle perturbation and a relevant induction in apoptosis.

Keywords: Acromegaly; Apoptosis; GH-secreting pituitary tumor; Long-term treatment; Somatostatin analogs.

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Conflict of interest statement

The authors declare that they have no conflict of interest.

Figures

Fig. 1
Fig. 1
Representative results of SST1 (222 bp), SST2 (249 bp) SST3 (256 bp), SST4 (240 bp) and SST5 (264 bp) mRNA expression, detected by TD-PCR, in GH3 and GH4C1 cell lines. PCR reactions contained the appropriate subtype-specific primers and water as a negative control. The quality of cDNA was confirmed by polymerase chain reaction of samples with primers for β-actin (A). L: Ladder
Fig. 2
Fig. 2
Effects of OCT (□) and PAS (■) on viability of GH3 (a, c) and GH4C1 (b, d) cell lines, as measured by MTS assay. Cells were incubated for 3 (a, b) and 6 days (c, d) without or with the drug at different concentrations (range 10–11–10–5 M). Dose–response curves represented best fit values of nonlinear regression (curve fit) of log (concentration drug) versus the percentage of vehicle-treated control (CTR). Values represent the mean and S.E.M. of at least three independent experiments in six replicates. *p < 0.05; **p < 0.01; ***p < 0.001 vs CTR
Fig. 3
Fig. 3
Cell cycle analysis after 6 days of incubation with OCT, PAS in GH3 (a–c) and GH4C1 (d–f) cell lines. Cells were detected by FACS analysis after staining with propidium iodide. Vehicle-treated control (CTR) values have been set to 100%. *p < 0.05; **p < 0.01 vs CTR
Fig. 4
Fig. 4
Modulation of cell death analysis after 6 days of incubation with OCT and PAS in GH3 (a–c) and GH4C1 (d–f) cell lines through flow cytometry with Annexin V and propidium iodine. The proportions of early (a, d), late (b, e) apoptotic, necrotic (c, f) cells are expressed as percentage compared with vehicle-treated control (CTR). Values represent the mean and SEM of at least three independent experiments. *p < 0.05; **p < 0.01; ***p < 0.001
Fig. 5
Fig. 5
Effect of SRLs on GH secretion in GH3 cell line. GH was measured by a rat/mouse GH ELISA (EMD Millipore, Billerica, Massachusetts) on cell culture media after 24 h (a) and 48 h (b) of incubation. GH values were normalized to the cellular proteins of each group. Results were expressed as a percentage compared with the vehicle-treated control (CTR) and represent the mean and SEM of at least three independent experiments. *p < 0.05 vs CTR
Fig. 6
Fig. 6
Effect of treatment with SRLs on GH-3 cells-induced angiogenesis. Representative epifluorescence images of 48 hpi Tg(fli1:EGFP)y1 zebrafish embryos injected with only PBS (a) or implanted with GH3 cells (b-g) and subsequently treated with DMSO vehicle (b and c), OCT (d and e) and PAS (f and g). The red channel was omitted in panels b, b′, d, d′, f and f′ to highlight the tumor-induced microvascular network. Digital magnifications of graft region are shown in white boxed regions b′, d′ and f′. The peritumoral density of endothelial structures, that sprouted from the SIV and CCV and reached the GH-3 tumor mass, did not result in difference in SRL-treated embryos compared to CTR. Here we show the quantification of tumor-induced endothelial structures at both 24 and 48 hpi (h). All images are oriented so that rostral is to the left and dorsal is at the top. Scale bar in a, 100 µm
Fig. 7
Fig. 7
Effect of treatment with SRLs on GH4C1 cells-induced angiogenesis. Representative epifluorescence images of 48 hpi Tg(fli1:EGFP)y1 zebrafish embryos injected with only PBS (a) or implanted with GH4C1 cells (b–g) and subsequently treated with DMSO vehicle (b and c), OCT (d and e) and PAS (f and g). The red channel was omitted in panels b, b′, d, d′, f and f′ to highlight the tumor-induced microvascular network. Digital magnifications of graft region are showed in white boxed regions b′, d′ and f′. The treatment with SRLs did not reduce the network density of endothelial structures, that sprouted from the SIV and CCV and reached the GH4C1 tumor mass, compared to vehicle-treated CTR embryos. Here we show the quantification of tumor-induced endothelial structures at both 24 and 48 hpi (h). All images are oriented so that the rostral is to the left and dorsal is at the top. Scale bar in a, 100 µm
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