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

posted on 20.12.2016 by Reuther C., Heinzle V., Nölting S., Herterich S., Hahner S., Halilovic E., Jeay S., Wuerthner J.U., Aristizabal Prada E.T., Spöttl G., Maurer J., Auernhammer C.J.

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.