Cabozantinib and Tivantinib, but Not INC280, Induce Antiproliferative and Antimigratory Effects in Human Neuroendocrine Tumor Cells in vitro: Evidence for 'Off-Target' Effects Not Mediated by c-Met Inhibition

Background/Aims: The hepatocyte growth factor/transmembrane tyrosine kinase receptor c-Met has been defined as a potential target in antitumoral treatment of various carcinomas. We aimed to investigate the direct effect of c-Met inhibition on neuroendocrine tumor cells in vitro. Methods: The effects of the multi-tyrosine kinase inhibitors cabozantinib and tivantinib and of the highly specific c-Met inhibitor INC280 were investigated in human pancreatic neuroendocrine BON1, bronchopulmonary NCI-H727 and midgut GOT1 cells in vitro. Results: INC280, cabozantinib and tivantinib inhibited c-Met phosphorylation, respectively. However, while equimolar concentrations (10 mu M) of cabozantinib and tivantinib inhibited cell viability and cell migration, INC280 had no inhibitory effect. Knockdown experiments with c-Met siRNA also did not demonstrate effects on cell viability. Cabozantinib and tivantinib caused a G2 arrest in neuroendocrine tumor cells. Conclusions: Our in vitro data suggest that c-Met inhibition alone is not sufficient to exert direct antitumoral or antimigratory effects in neuroendocrine tumor cells. The multi-tyrosine kinase inhibitors cabozantinib and tivantinib show promising antitumoral and antimigratory effects in neuroendocrine tumor cells, which are most probably 'off-target' effects, not mediated by c-Met. (C) 2015 S. Karger AG, Base

Additive Anti-Tumor Effects of Lovastatin and Everolimus In Vitro through Simultaneous Inhibition of Signaling Pathways

This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are creditedThis work was supported by a research grant from the Ludwig-Maximilians University of Munich (Förderprogramm für Forschung und Lehre [FöFoLe], grant number 865/829)

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 p53wildtype neuroendocrine tumor cell line GOT1

Background/aims: 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 p53wild 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. Methods: 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. Results: While p53wild type GOT1 cells were sensitive to NVP-CGM097, p53mutated BON1 and p53mutated 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% (p < 0.05), 77.4 ± 6.6% (p < 0.01), and 47.7 ± 9.2% (p < 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. Conclusions: MDM2 inhibition seems a promising novel therapeutic target in neuroendocrine tumors harboring p53wild type. Further investigations should examine the potential role of MDM2 inhibitors in neuroendocrine tumor treatment. Keywords: 5-Fluorouracil; Everolimus; GOT1; MDM2 inhibitor; NVP-CGM097; Neuroendocrine tumor; Temozolomide; p21; p53

Cell viability in HepG2 cells: HepG2 cells were pre-treated with lovastatin for 24h before everolimus was added, and the combination of both drugs was incubated for 48h (A) or 120h (B).

<p>The single drugs were incubated for the same time as in combination. The mean percentage of cell viability, relative to the untreated control, ± SEM (error bars) is shown. (A) Treatment with 5–20 μM lovastatin or with 10 nM everolimus alone significantly decreased HepG2 cell viability. Combination treatment with 10 μM lovastatin and 10 nM everolimus significantly more strongly reduced cell viability than each drug separately. (B) Treatment with 5–20 μM lovastatin or with 10 nM everolimus separately significantly decreased HepG2 cell viability. Combination treatment with 10–20 μM lovastatin and 10 nM everolimus significantly more strongly reduced cell viability compared to each drug given alone. * P ≤ 0.05, ** P ≤ 0.01, *** P ≤ 0.001 (compared to the control); @ P ≤ 0.05, @@ P ≤ 0.01, @@@ P ≤ 0.001 (combination compared to each drug separately).</p

Signaling pathways in BON1, H727, HepG2 and Huh7 cells.

<p>Effects of 48h combination treatment with 20 μM lovastatin and 10 nM everolimus after 24h pre-treatment with lovastatin on signaling pathways in BON1, H727, HepG2 and Huh7 cells compared to treatment with each drug separately. Data are shown as mean percentage of the absolute phospho-protein expression, relative to the untreated control, ± SEM.</p

Cell viability in MPC (A) and MTT (B) cells: MPC and MTT cells were pre-treated with lovastatin for 24h before everolimus was added, and the combination of both drugs was incubated for 48h.

<p>The single drugs were incubated for the same time as in combination. The mean percentage of cell viability, relative to the untreated control, ± SEM (error bars) is shown. Treatment with 10 μM lovastatin or with 10 nM everolimus separately significantly reduced MPC and MTT cell viability, compared to the control. 48h combination treatment with 10 μM lovastatin and 10 nM everolimus significantly more potently decreased MPC and MTT cell viability compared to each drug given separately. * P ≤ 0.05, ** P ≤ 0.01, *** P ≤ 0.001 (compared to the control); @ P ≤ 0.05, @@ P ≤ 0.01, @@@ P ≤ 0.001 (combination compared to each drug separately).</p

Cell viability in H727 cells: H727 cells were pre-treated with lovastatin for 24h before everolimus was added, and the combination of both drugs was incubated for 48h (A) or 120h (B).

<p>The single drugs were incubated for the same time as in combination. The mean percentage of cell viability, relative to the untreated control, ± SEM (error bars) is shown. (A) Treatment with 5–20 μM lovastatin or with 10 nM everolimus alone significantly decreased H727 cell viability. Combination treatment with 20 μM lovastatin and 10 nM everolimus significantly more strongly reduced cell viability than each drug separately. (B) Treatment with 5–20 μM lovastatin or with 10 nM everolimus separately significantly reduced H727 cell viability. The combination of 5–20 μM lovastatin with 10 nM everolimus significantly more strongly reduced cell viability compared to each drug given separately. * P ≤ 0.05, ** P ≤ 0.01, *** P ≤ 0.001 (compared to the control); @ P ≤ 0.05, @@ P ≤ 0.01, @@@ P ≤ 0.001 (combination compared to each drug separately).</p