Supplementary MaterialsS1 Fig: Uncropped western blots. GUID:?965B985D-5899-44AD-8C7A-2ACC0955A053 S20 Fig: Uncropped western blots. (TIF) pone.0182852.s020.tif (2.4M) GUID:?7237DD40-CD61-4378-81A2-AAA760E33501 S21 Fig: Uncropped western blots. (TIF) pone.0182852.s021.tif (3.4M) GUID:?A40F494C-2D39-438D-A64B-31B911DEF034 S22 Fig: Uncropped western blots. (TIF) pone.0182852.s022.tif (3.2M) GUID:?5DEE7A65-586B-49DB-9E2F-8835D77BD54E S23 Fig: Uncropped western blots. (TIF) pone.0182852.s023.tif (2.6M) GUID:?9803932B-FAC5-464C-BAB1-457B8082A174 S24 Fig: Western blot 1 M and higher concentrations of BYL719 pGSK3 and pIGF1R. (TIF) pone.0182852.s024.tif (487K) GUID:?03711A5C-0315-487E-9D5D-E4F0FC27501D S25 Fig: Western blot 2.5 M and higher concentrations of BYL719 pGSK3 and pIGF1R. (TIF) pone.0182852.s025.tif (463K) GUID:?E8636DA2-1D41-405C-A570-21AF5E9977AC S26 Fig: Western blot NVP-AEW541 plus BYL719 without IGF1 stimulation. (TIF) pone.0182852.s026.tif (1.0M) GUID:?F5117D4B-7EAB-46F8-9EB1-B6345FB13BBD S27 Fig: Western blot NVP-AEW541 plus BYL719 with IGF1 stimulation. (TIFF) pone.0182852.s027.tiff (1.3M) GUID:?1B07AAAC-D296-4045-B9D6-1A06711BD502 S28 Fig: Comparison of the effects of BYL719 versus BKM120 on BON-1 cell viability. (TIF) pone.0182852.s028.tif (364K) GUID:?8572F1DE-F28A-4DDD-BE38-6E402D28F22A S29 Fig: Uncropped western blots. (TIF) pone.0182852.s029.tif (559K) GUID:?1FFC3D83-A47D-47D9-9195-45E209C3CBC6 S30 Fig: Uncropped western blots. (TIF) pone.0182852.s030.tif (1.1M) GUID:?F11ADB10-9F7B-4B6C-99CA-6E42F75F4B2A S1 Table: Densitometry analysis of the performed western blots. Band intensities were quantified from at least 3 independent experiments for each cell line and protein, and are expressed as the mean percentage relative to the untreated control (100%). The means and standard deviations are reported as geometric means and geometric standard deviations of the relative increase, respectively. A geometric mean of “1.0” has to be interpreted Sacubitrilat as “equal to the control group” and for the geometric standard deviation 1.0 refers to no variation. Statistically significantly different results in comparison to the control are shown as p-values, considering p 0.05 as significant.(XLSX) pone.0182852.s031.xlsx (27K) GUID:?A994E5FE-5003-4B40-88E0-3E52C082AE3C Data Availability StatementAll relevant data are within the paper and its Supporting Information files. Abstract Background/Aims The therapeutic options for metastatic neuroendocrine tumors Sacubitrilat (NETs) are limited. As PI3K signaling is often activated in NETs, we have assessed the effects of selective PI3Kp110 inhibition by the novel agent BYL719 on cell viability, colony formation, apoptosis, cell cycle, signaling pathways, differentiation and secretion in pancreatic (BON-1, QGP-1) and pulmonary (H727) NET cell lines. Methods Cell viability was investigated by WST-1 assay, colony formation by clonogenic assay, apoptosis by caspase3/7 assay, the cell cycle by FACS, cell signaling by Western blot analysis, expression of chromogranin A and somatostatin receptors 1/2/5 by RT-qPCR, and chromogranin A secretion by ELISA. Results BYL719 dose-dependently decreased cell viability and colony formation with the highest sensitivity in BON-1, followed by H727, and lowest sensitivity in QGP-1 cells. BYL719 induced apoptosis and G0/G1 cell cycle arrest associated with increased p27 expression. Western blots showed inhibition of Sacubitrilat PI3K downstream targets to a varying degree in the different cell lines, but IGF1R activation. The most sensitive BON-1 cells displayed a significant, and H727 cells a non-significant, GSK3 Rabbit Polyclonal to Syndecan4 inhibition after BYL719 treatment, but these effects do not appear to be mediated through the IGF1R. In contrast, the most resistant QGP-1 cells showed no GSK3 inhibition, but a modest geneis activated by different receptor tyrosine kinases (such as IGFR, EGFR, VEGFR, FGFR, RET) and in turn activates AKT which leads to inhibition of TSC1/2 and consequently to disinhibition/activation of and in clinical studies [1, 6], and has been approved for the treatment of pancreatic [5] and, very recently, of gastrointestinal and lung NETs [3, 4]. However, mTORC1 inhibition leads to a compensatory activation of PI3K/AKT signaling via p70S6K and IRS-1 activation, associated with an increase in pAKTT308 (via PI3K/PDK1), pAKTS473 (via mTORC2) and RAS/RAF/MEK/ERK signaling [15C19] (Fig 1). This compensatory up-regulation of PI3K/AKT and RAS/RAF/MEK/ERK may cause tumor cell resistance of initially sensitive cells [18C26]. Thus, it could be speculated that PI3K inhibitors working more upstream may bypass this mechanism of resistance and be more effective than Sacubitrilat mTORC1-inhibition alone. Indeed, different panPI3K inhibitors (LY294002, BKM120) or the dual PI3K/mTORC1/2 inhibitor BEZ235 alone and in combination with mTORC1 inhibitors and a MEK inhibitor, respectively, have shown anti-tumor potential in NET cells and [17, 18, 27C31]. In pancreatic NET cells, a synergistic effect of everolimus plus BEZ235 has been reported [18]. However, global clinical development of BEZ235the most effective agent in pancreatic NET cells [18]has been terminated, and two clinical trials in pancreatic NETs were stopped early due to poor tolerability, frequent treatment discontinuation, a non-fulfilled statistical endpoint and, moreover, no clear superiority to.