High level MycN expression in non-MYCN amplified neuroblastoma is induced by the combination treatment nutlin-3 and doxorubicin and enhances chemosensitivity
Department of Pediatrics, Division of Hematology and Oncology, Emory University School of Medicine, Aflac Cancer Center and Blood Service, Atlanta, GA 30322, USA.Oncology Reports (Impact Factor: 2.3). 12/2009; 22(6):1443-9. DOI: 10.3892/or_00000586
MYCN gene amplification is a negative prognostic indicator in neuroblastoma and high level MycN expression in Stage IV neuroblastoma is generally a hallmark of poor patient outcome. However, high level expression of the MycN protein in neuroblastoma cells lacking MYCN amplification suppresses growth and drives apoptosis; this, in part, explains the absence of clinical observations of high level MycN in neuroblastoma lacking MYCN amplification. In the current study, we found that combination treatment with nutlin-3 and doxorubicin upregulated MycN expression in non-MYCN-amplified neuroblastoma cells at both the protein and mRNA levels. The induced expression of MycN in non-MYCN-amplified cells inhibited cell proliferation and increased apoptosis. MycN induction also upregulated p53, p21 and Bax protein levels, as well as mRNA levels for the positive neuroblastoma prognostic factors CD44 and EFNB3. Blocking MycN reversed these effects. These results were corroborated by findings using a MycN-inducible system in SHEP cells, another MYCN non-amplified neuroblastoma cell line. Our results indicate that doxorubicin/nutlin-3 combination treatment both induces expression of MycN in a non-MYCN-amplified background and sensitizes neuroblastoma cells to chemotherapy. These findings support the idea that induction of MycN in non-MYCN-amplified cells drives neuroblastoma cells toward apoptosis and suggest that combination nutlin-3/doxorubicin treatment may be clinically important.
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ABSTRACT: Chemotherapy to date has not been effective in the treatment of cholangiocarcinoma. However, gemcitabine, a novel nucleoside analog, has shown remarkable biological activity against cholangiocarcinoma in some clinical studies. Combinations of gemcitabine with other agents have also shown promising results, with a tolerable toxicity profile. Nutlin-3 is a small-molecule inhibitor that acts to inhibit murine double minute-2 (MDM2) binding to p53 or p73 and subsequently activates p53- or p73-dependent apoptosis signaling pathways. To investigate their effects in combination, a p53-mutant cholangiocarcinoma line HuCCT1 was treated with Nutlin-3 and/or gemcitabine in the current study. Cell proliferation assay, apoptosis assay, Western blot, coimmunoprecipitation, and small interfering RNA (siRNA) experiments were analyzed in HuCCT1 cells. Antitumoral effects of Nutlin-3 and/or gemcitabine targeting the p73/MDM2 pathways were evaluated. Nutlin-3 and gemcitabine can both inhibit the growth of HuCCT1 cells. Nutlin-3 induces apoptosis and potentiates the cytotoxic effect of gemcitabine through disrupting the binding of p73 with MDM2. Nutlin-3 leads to activation of caspases, increase levels of puma and bax, and decrease the expression of bcl-2. Blocking p73 function with a siRNA attenuates the apoptosis induced by gemcitabine, Nutlin-3, and gemcitabine/Nutlin-3 combination. Our data provide evidence that p73 might compensate for p53 function in gemcitabine-induced apoptosis of HuCCT1 cells. Nutlin-3 acts through the inhibition of p73-MDM2 with subsequent activation of the apoptotic pathway signaling, which leads to the increase in chemosensitivity to gemcitabine. In summary, our findings suggest that Nutlin-3 will be active in the treatment of p53-mutant cholangiocarcinoma, especially when in combination with gemcitabine.
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ABSTRACT: The aims of this study were to define the mRNA expression profiles of MYCN, DDX1, TrkA, and TrkC in biopsy tumor samples from 64 Brazilian patients with neuroblastomas of different risk stages and to correlate altered expression with prognostic values. Patients were retrospectively classified into low- (n = 11), intermediate- (n = 18), and high-risk (n = 35) groups using standard criteria. The mRNA levels of the above genes were measured by quantitative real-time polymerase chain reaction. Univariate analyses were performed and survival curves were plotted by the Kaplan-Meier method. Of the 64 patients, 53% were female and 62.5% were older than 18 months. The 5-year overall survival (OS) for the entire cohort was 40.3%, with inferior median OS in patients identified in the intermediate- and high-risk groups. A significant difference in OS with respect to TrkA mRNA expression was found for the high-risk group vs. either the low- or intermediate-risk groups (P < 0.01, log rank test). Within the intermediate-risk group, neuroblastoma patients with positive TrkA mRNA expression had better clinical outcomes than patients with no TrkA transcript expression (P = 0.004). Another difference in OS was only found between the intermediate- and high-risk groups (P < 0.027, log rank test). No significant correlation of mRNA expression and survival outcome could be detected for the MYCN, DDX1. Positive expression of TrkA mRNA may be a clinically useful addition to the current risk classification system, allowing the identification of NB tumors with favorable prognosis.
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ABSTRACT: Nutlin-3 is a small molecule inhibitor of the MDM2/p53 interaction, which leads to the non-genotoxic p53 stabilization, activation of cell cycle arrest and apoptosis pathways. A series of recent studies have strengthened the concept that selective, non-genotoxic p53 activation by Nutlin-3 might represent an alternative to the current cytotoxic chemotherapy, in particular for pediatric tumors and for hematological malignancies, which retain a high percentage of p53(wild-type) status at diagnosis. Like most other drugs employed in cancer therapy, it will be unlikely that Nutlin-3 will be used as a monotherapy. In this respect, Nutlin-3 shows a synergistic cytotoxic effect when used in combination with innovative drugs, such as TRAIL or bortozemib. Although Nutlin-3 is currently in phase I clinical trial for the treatment of retinoblastoma, its effects on normal tissues and cell types remain largely to be determined and will require further investigation in the future years.
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