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<p>Somatostatin analogs (SSA) are well-established antisecretory drugs in functionally active neuroendocrine tumors (NET). Two placebo-controlled trials have recently demonstrated significant improvement of progression-free survival under SSA treatment. Furthermore, somatostatin receptor (SSTR) overexpression in NET has also been utilized for diagnostic imaging and peptide receptor radionuclide therapy (PRRT). However, PRRT in NET is associated mostly with partial and minor remission, while other radionuclide therapies reach complete remissions in up to 75% of cases. This study assessed a potential radiosensitizing effect of SSA treatment in five established NET cell line models: BON, QGP-1, LCC-18, H727, and UMC-11. Irradiation was found to significantly inhibit proliferation, while no additional effect by octreotide treatment was observed. Intriguingly, no impact of SSA treatment alone was found in any of these NET cell lines when systematically analyzing cell viability, proliferation, and cell cycle distribution. Investigation of the causes for this octreotide resistance led to demonstration of low octreotide binding and scarce SSTR, specifically SSTR2 expression as compared to levels found in human NETs. The resistance toward SSA treatment in viability and proliferation assays could not be overcome by re-expression of SSTR2 in two of the cell lines. These results provide systematic evidence for a lack of authentic, tumor-like SSTR expression, and function in five frequently used NET cell line models and point to the need for more physiologic tumor model systems.</p

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<p>Somatostatin analogs (SSA) are well-established antisecretory drugs in functionally active neuroendocrine tumors (NET). Two placebo-controlled trials have recently demonstrated significant improvement of progression-free survival under SSA treatment. Furthermore, somatostatin receptor (SSTR) overexpression in NET has also been utilized for diagnostic imaging and peptide receptor radionuclide therapy (PRRT). However, PRRT in NET is associated mostly with partial and minor remission, while other radionuclide therapies reach complete remissions in up to 75% of cases. This study assessed a potential radiosensitizing effect of SSA treatment in five established NET cell line models: BON, QGP-1, LCC-18, H727, and UMC-11. Irradiation was found to significantly inhibit proliferation, while no additional effect by octreotide treatment was observed. Intriguingly, no impact of SSA treatment alone was found in any of these NET cell lines when systematically analyzing cell viability, proliferation, and cell cycle distribution. Investigation of the causes for this octreotide resistance led to demonstration of low octreotide binding and scarce SSTR, specifically SSTR2 expression as compared to levels found in human NETs. The resistance toward SSA treatment in viability and proliferation assays could not be overcome by re-expression of SSTR2 in two of the cell lines. These results provide systematic evidence for a lack of authentic, tumor-like SSTR expression, and function in five frequently used NET cell line models and point to the need for more physiologic tumor model systems.</p

Urolinin: The First Linear Peptidic Urotensin-II Receptor Agonist

This study investigated the role of individual U-II amino acid positions and side chain characteristics important for U-IIR activation. A complete permutation library of 209 U-II variants was studied in an activity screen that contained single substitution variants of each position with one of the other 19 proteinogenic amino acids. Receptor activation was measured using a cell-based high-throughput fluorescence calcium mobilization assay. We generated the first complete U-II substitution map for U-II receptor activation, resulting in a detailed view into the structural features required for receptor activation, accompanied by complementary information from receptor modeling and ligand docking studies. On the basis of the systematic SAR study of U-II, we created 33 further short and linear U-II variants from eight to three amino acids in length, including d- and other non-natural amino acids. We identified the first high-potency linear U-II analogues. Urolinin, a linear U-II agonist (nWWK-Tyr­(3-NO₂)-Abu), shows low nanomolar potency as well as improved metabolic stability

Representative western blots of the investigated signaling pathways, apoptosis and cell cycle markers in BON-1, QGP-1 and H727 cells: The means, standard deviations and p-values of the western blot quantification and statistical analysis from at least three independent replicates are given in S1 Table.

<p>Representative western blots of the investigated signaling pathways, apoptosis and cell cycle markers in BON-1, QGP-1 and H727 cells: The means, standard deviations and p-values of the western blot quantification and statistical analysis from at least three independent replicates are given in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0182852#pone.0182852.s031" target="_blank">S1 Table</a>.</p

Neuroendocrine marker expression was increased by BYL719 treatment.

<p>A: BON-1, H727 and QGP-1 cells were treated with 10 μM BYL719 for 96 h and mRNA expression of Chromogranin A <i>(CHGA)</i>, <i>SSTR1</i>, <i>SSTR2</i> and <i>SSTR5</i> was analyzed by qPCR. B: BON-1, H727 and QGP-1 cells were treated with 1 μM BYL719 for 96 h and mRNA expression of <i>SSTR2</i> was analyzed by qPCR; Values are summarized from three independent experiments of three replicates per data point. * p<0.05; ** p≤0.01; *** p≤0.001.</p

CgA secretion of NET cells was not altered by BYL719 treatment.

<p>BON-1, H727 and QGP-1 cells were treated with vehicle, PDBu (positive control) or BYL719 (1 μM, 10 μM) for 24 h; the supernatant was collected for CgA measurements (two independent experiments with three replicates per data point).</p

Cell lines were treated with BYL719 (IC₅₀ and IC₈₅) for 72 h.

<p>Cells were stained with PI (DNA content) analysis solely included and mitosis-specific Phospho(Ser10)-Histone H3 immunostain followed by flow cytometry assessment (single cells). According to the stain, events were divided into cells of several cell cycle phases or were classified as sub-G1-events indicating cell death (FlowJo software), given in percent of all detected cells (mean ± SD) of at least four independent experiments. A: Cell cycle. B: Mitotic index. * p<0.05; ** p≤0.01; *** p≤0.001; **** p≤0.0001.</p

Assessment of apoptosis: Caspase 3/7 assay results after 24 h treatment with different doses of BYL719 are shown as mean percentage of caspase 3/7 activity referred to the untreated control (100%) ± SD: Strongest apoptosis was induced in BON-1 cells, followed by H727 cells.

<p>QGP-1 cells showed the least but still significant apoptosis. * p<0.05; ** p≤0.01; *** p≤0.001 (two independent experiments with three replicates per data point).</p

The combination therapy with BYL719 plus everolimus strongly induced SSTR2 expression in BON-1 and QGP-1 cells.

<p>Cells have been treated with 5 μM BYL719, 5 nM everolimus, or the combination of both for 48h (A) or with 1 μM BYL719, 1 nM everolimus or the combination of both for 96h (B). SSTR2 expression was analyzed by Western Blot (A) and qPCR (B). Representative western blot of two replicates; qPCR results are summarized from three independent experiments of three replicates. * p<0.05; ** p≤0.01; *** p≤0.001.</p