Erratum: The Novel mTOR Inhibitor RAD001 (Everolimus) Induces Antiproliferative Effects in Human Pancreatic Neuroendocrine Tumor Cells

<i>Background/Aim:</i> Tumors exhibiting constitutively activated PI(3)K/Akt/mTOR signaling are hypersensitive to mTOR inhibitors such as RAD001 (everolimus) which is presently being investigated in clinical phase II trials in various tumor entities, including neuroendocrine tumors (NETs). However, no preclinical data about the effects of RAD001 on NET cells have been published. In this study, we aimed to evaluate the effects of RAD001 on BON cells, a human pancreatic NET cell line that exhibits constitutively activated PI(3)K/Akt/mTOR signaling. <i>Methods:</i> BON cells were treated with different concentrations of RAD001 to analyze its effect on cell growth using proliferation assays. Apoptosis was examined by Western blot analysis of caspase-3/PARP cleavage and by FACS analysis of DNA fragmentation. <i>Results:</i> RAD001 potently inhibited BON cell growth in a dose-dependent manner which was dependent on the serum concentration in the medium. RAD001-induced growth inhibition involved G0/G1-phase arrest as well as induction of apoptosis. <i>Conclusion:</i> In summary, our data demonstrate antiproliferative and apoptotic effects of RAD001 in NET cells in vitro supporting its clinical use in current phase II trials in NET patients

Supplementary Material for: GSK3α/β: A Novel Therapeutic Target for Neuroendocrine Tumors?

<p><b><i>Introduction:</i></b> Glycogen synthase kinase 3α/β (GSK3α/β) is a serine/threonine kinase that plays a critical role in cancer. <b><i>Aims:</i></b> In this study, we evaluated the effects of the specific GSK3α/β inhibitor AR-A014418 in vitro to gain novel insights into GSK3α/β signaling in neuroendocrine tumors (NETs). <b><i>Materials and Methods:</i></b> Human NET cell lines (BON1, QGP1, H727, and GOT1) were treated with different concentrations of AR-A014418 alone and in combination with lovastatin, everolimus, 5-fluorouracil (5-FU), and γ-irradiation. <b><i>Results:</i></b> AR-A014418 significantly dose- and time-dependently decreased cell viability in all 4 NET cell lines through inhibition of epithelial growth factor receptor and mTORC1/p70S6K signaling, as well as cyclin D3 downregulation and induction of pChk1. In all cell lines tested, FACS analysis showed an AR-A014418-induced increase in the sub-G1 phase, reflecting cell death. Apoptosis induction was observed in H727, GOT1 and QGP1 cells, but not in BON1 cells. Furthermore, significant antimigratory effects upon GSK3α/β inhibition were found and were associated with β-catenin downregulation in all cell lines tested. Compensatory upregulation of pAkt and pERK in response to GSK3α/β inhibition was prevented by combining AR-A014418 with the ERK and Akt inhibitor lovastatin. Accordingly, the lovastatin/AR-A014418 combination was synergistic in BON1 and QGP1 cells. Moreover, AR-A014418 displayed promising chemosensitizing effects on 5-FU in QGP1 and slight radiosensitizing properties in BON1 and QGP1 cells. <b><i>Conclusion:</i></b> Our data provide new insights into the role of GSK3α/β in NETs and suggest that GSK3α/β inhibition could be a novel therapeutic option in NETs, especially in combination with lovastatin or 5-FU, depending on tumor entity.</p

Supplementary Material for: The HDM2 (MDM2) Inhibitor NVP-CGM097 Inhibits Tumor Cell Proliferation and Shows Additive Effects with 5-Fluorouracil on the p53-p21-Rb-E2F1 Cascade in the p53wild type Neuroendocrine Tumor Cell Line GOT1

<p><b><i>Background/Aims:</i></b> The tumor suppressor p53 is depleted in many tumor cells by the E3 ubiquitin ligase mouse double minute 2 homolog (MDM2) through MDM2/p53 interaction. A novel target for inhibiting p53 degradation and for causing reexpression of p53^{wild type} is inhibition of MDM2. The small molecule NVP-CGM097 is a novel MDM2 inhibitor. We investigated MDM2 inhibition as a target in neuroendocrine tumor cells in vitro. <b><i>Methods:</i></b> Human neuroendocrine tumor cell lines from the pancreas (BON1), lung (NCI-H727), and midgut (GOT1) were incubated with the MDM2 inhibitor NVP-CGM097 (Novartis) at concentrations from 4 to 2,500 nM. <b><i>Results:</i></b> While p53^{wild type} GOT1 cells were sensitive to NVP-CGM097, p53^mutated BON1 and p53^mutated NCI-H727 cells were resistant to NVP-CGM097. Incubation of GOT1 cells with NVP-CGM097 at 100, 500, and 2,500 nM for 96 h caused a significant decline in cell viability to 84.9 ± 9.2% (<i>p</i> < 0.05), 77.4 ± 6.6% (<i>p</i> < 0.01), and 47.7 ± 9.2% (<i>p</i> < 0.01). In a Western blot analysis of GOT1 cells, NVP-CGM097 caused a dose-dependent increase in the expression of p53 and p21 tumor suppressor proteins and a decrease in phospho-Rb and E2F1. Experiments of co-incubation of NVP-CGM097 with 5-fluorouracil, temozolomide, or everolimus each showed additive antiproliferative effects in GOT1 cells. NVP-CGM097 and 5-fluorouracil increased p53 and p21 expression in an additive manner. <b><i>Conclusions:</i></b> MDM2 inhibition seems a promising novel therapeutic target in neuroendocrine tumors harboring p53^{wild type}. Further investigations should examine the potential role of MDM2 inhibitors in neuroendocrine tumor treatment.</p