[PMC free article] [PubMed] [Google Scholar] 45. NB patients. on chronic lymphocytic leukemia and resulted in severe cytotoxicities to normal tissue . And despite proteasome inhibitors being highly active, resistance is commonly observed [31, 32]. Therefore, novel therapeutic brokers with improved efficacy need to be developed. Carfilzomib (CFZ) is usually a novel proteasome inhibitor that has already been approved by the FDA for treating the relapsed and refractory multiple myeloma in July of 2012 [33C36]. Prior studies exhibited that CFZ irreversibly inhibits 26S proteasome activity and efficiently stabilize IB by inhibiting its degradation, subsequently inhibiting NF-B activation and inducing apoptosis pathway [37C39]. In addition, CFZ activated the users of MAPK family, including the stress-activated kinases p38, JNK, and ERK1/2 in leukemia/lymphoma, lung malignancy , etc. Herein, we evaluate the cytotoxic effects of CFZ on NB cells. Our results demonstrate that CFZ induced apoptosis and enhanced doxorubicin (Dox)-induced apoptosis through inhibiting the NF-B activation and activating p38 and JNK pathway in NB. Our studies suggest that novel proteasome inhibitor CFZ might be a potential therapeutic agent for NB patients. RESULTS Proteasome inhibitor CFZ exhibits cytotoxic effect on NB cells To determine the potential cytotoxic effect of CFZ on NB cells, the CCK-8 assay was measured on six NB cell lines, including three N-myc amplified cell lines (IMR-32, NB-19, NGP) and three N-myc non-amplified cell lines (LA-N-6, SH-SY5Y, SK-N-AS) (Physique ?(Figure1).1). The cell viabilities of all cell lines tested were greatly reduced with increasing concentrations of CFZ after being treated for 72 h (Physique ?(Figure1A).1A). The IC50s of CFZ in all six cell lines were calculated, ranged from 3.31 nM to 48.64 nM (Figure ?(Figure1A).1A). Our results indicate that CFZ inhibits cell growth in a dose-dependent manner in NB cell lines. In addition, the cytotoxic effect of CFZ in NB cells was further confirmed by cell morphology switch after the treatment (Physique ?(Figure1B1B). Open in a separate window Physique 1 CFZ shows cytotoxic effect on a panel of six NB cellsA. Six NB cell lines were treated with increasing concentrations (0 M, 0.005 M, 0.01 M, 0.02 M, 0.05 M, 0.1 M, 0.2 M, 0.5 M, 1 M, 2 M, 5 M, 10 M, 20 M) of CFZ for 72 h. Cell viability was assessed by CCK-8 assay. data, CFZ treatment caused Caspase 3 cleavage in tumor cells KPT-9274 from CFZ treatment group (Physique ?(Figure6A).6A). In addition, owing that some N-myc amplified cell lines are more resistant to CFZ IMPG1 antibody treatment compared to N-myc non-amplified cells, we tested whether CFZ could enhance the cytotoxic effects of Dox in N-myc amplified tumors and were used to determine the statistical significance among drug treatment group and control group. Each assay was repeated at least twice, and representative results were presented. KPT-9274 and selective antitumor activity of a KPT-9274 novel orally bioavailable proteasome inhibitor MLN9708 against multiple myeloma cells. Clinical malignancy research. 2011;17:5311C5321. [PMC KPT-9274 free article] [PubMed] [Google Scholar] 29. Garcia-Gomez A, Quwaider D, Canavese M, Ocio EM, Tian Z, Blanco JF, Berger AJ, Ortiz-de-Solorzano C, Hernandez-Iglesias T, Martens AC, Groen RW, Mateo-Urdiales J, Fraile S, et al. Preclinical activity of the oral proteasome inhibitor MLN9708 in Myeloma bone disease. Clinical malignancy research. 2014;20:1542C1554. [PubMed] [Google Scholar] 30. Gupta SV, Hertlein E, Lu Y, Sass EJ, Lapalombella R, Chen TL, Davis ME, Woyach JA, Lehman A, Jarjoura D, Byrd JC, Lucas DM. The proteasome inhibitor carfilzomib functions independently of p53 to induce cytotoxicity and an atypical NF-kappaB response in chronic lymphocytic leukemia cells. Clinical malignancy research. 2013;19:2406C2419. [PMC free article] [PubMed] [Google Scholar] 31. Levin N, Spencer A, Harrison SJ, Chauhan D, Burrows FJ, Anderson KC, Reich.