The Novel SSTR3 Agonist ITF2984 Exerts Antimitotic and Proapoptotic Effects in Human Non-Functioning Pituitary Neuroendocrine Tumor (NF-PitNET) Cells

Abstract

Somatostatin receptor ligands (SRLs) with high affinity for somatostatin receptors 2 and 5 (SSTR2 and SSTR5) are poorly efficacious in NF-PitNETs, expressing high levels of SSTR3. ITF2984 is a pan-SSTR ligand with high affinity for SSTR3, able to induce SSTR3 activation and to exert antitumoral activity in the MENX rat model. The aim of this study was to test ITF2984's antiproliferative and proapoptotic effects in NF-PitNET primary cultured cells derived from surgically removed human tumors and to characterize their SSTR expression profile. We treated cells derived from 23 NF-PitNETs with ITF2984, and a subset of them with octreotide, pasireotide (SRLs with high affinity for SSTR2 or 5, respectively), or cabergoline (DRD2 agonist) and we measured cell proliferation and apoptosis. SSTR3, SSTR2, and SSTR5 expression in tumor tissues was analyzed by qRT-PCR and Western blot. We demonstrated that ITF2984 reduced cell proliferation (-40.8 (17.08)%, p < 0.001 vs. basal, n = 19 NF-PitNETs) and increased cell apoptosis (+41.4 (22.1)%, p < 0.001 vs. basal, n = 17 NF-PitNETs) in all tumors tested, whereas the other drugs were only effective in some tumors. In our model, SSTR3 expression levels did not correlate with ITF2984 antiproliferative nor proapoptotic effects. In conclusion, our data support a possible use of ITF2984 in the pharmacological treatment of NF-PitNET.

Keywords: ITF2984; NF-PitNETs; SSTRs.

Figure 1

ITF2984 effects on cell proliferation in primary cultured non-functioning pituitary neuroendocrine (NF-PitNET) cells. NF-PitNET cells were incubated for 72 h at 37 °C with increasing concentrations of ITF2984 (a) or with 100 nM ITF2984 (n = 8), 100 nM pasireotide (n = 6), 100 nM octreotide (n = 5), and 100 nM cabergoline (n = 8) (b). BrdU incorporation in newly synthesized DNA was measured. Each determination was carried out in triplicate. The graphs show the median (interquartile range (IQR)). *** p < 0.001 vs. corresponding basal. Pas = pasireotide, Octr = octreotide, Cab = cabergoline.

Figure 2

ITF2984 effects on cell apoptosis in primary cultured NF-PitNET cells. NF-PitNET cells were incubated for 48 h at 37 °C with increasing concentrations of ITF2984 (a), or with 100 nM ITF2984 (n = 10), 100 nM pasireotide (n = 7), 100 nM octreotide (n = 7), and 100 nM cabergoline (n = 10) (b). Caspase 3/7 activity was measured. Each determination was carried out in triplicate. The graphs show the median (IQR). ** p < 0.01; *** p < 0.001 vs. corresponding basal. Pas = pasireotide, Octr = octreotide, Cab = cabergoline.

Figure 3

(a) Graphs show the expression levels of somatostatin receptor (SSTR)-type 3, 2, and 5 transcripts measured by RT-qPCR in NF-PitNET tissues. In the upper graph, medians are shown (horizontal bar). The lower graph shows the expression levels of the three receptors in each NF-PitNET sample. 1–23 = NF-PitNET samples. * p < 0.05. (b) Protein expression of SSTR3, SSTR2, and SSTR5 normalized to GAPDH and to internal positive control. Proteins extracted from each tissue sample were analyzed by Western blotting and quantified by densitometric analysis. 2–23 = NF-PitNET samples. (c) Graph shows Spearman correlation between mRNA levels of SSTR3 and SSTR2 or SSTR3 and SSTR5.

Figure 3

(a) Graphs show the expression levels of somatostatin receptor (SSTR)-type 3, 2, and 5 transcripts measured by RT-qPCR in NF-PitNET tissues. In the upper graph, medians are shown (horizontal bar). The lower graph shows the expression levels of the three receptors in each NF-PitNET sample. 1–23 = NF-PitNET samples. * p < 0.05. (b) Protein expression of SSTR3, SSTR2, and SSTR5 normalized to GAPDH and to internal positive control. Proteins extracted from each tissue sample were analyzed by Western blotting and quantified by densitometric analysis. 2–23 = NF-PitNET samples. (c) Graph shows Spearman correlation between mRNA levels of SSTR3 and SSTR2 or SSTR3 and SSTR5.