Ambrosini G, Sambol EB, Carvajal D, Vassilev LT, Singer S, Schwartz GK.. Mouse Double minute antagonist Nutlin-3a enhances chemotherapy-induced apoptosis in cancer cells with mutant p53 by activating E2F1. Oncogene 24: 3473-3481

Laboratory of New Drug Development, Department of Medicine, Memorial Sloan-Kettering Cancer Center, New York, NY 10021, USA.
Oncogene (Impact Factor: 8.46). 06/2007; 26(24):3473-81. DOI: 10.1038/sj.onc.1210136
Source: PubMed


MDM2 is a critical negative regulator of the p53 tumor suppressor protein. Recently, small-molecule antagonists of MDM2, the Nutlins, have been developed to inhibit the p53-MDM2 interaction and activate p53 signaling. However, half of human cancers have mutated p53 and they are resistant to Nutlin treatment. Here, we report that treatment of the p53-mutant malignant peripheral nerve sheath (MPNST) and p53-null HCT116 cells with cisplatin (Cis) and Nutlin-3a induced a degree of apoptosis that was significantly greater than either drug alone. Nutlin-3a also increased the cytotoxicity of both carboplatin and doxorubicin in a series of p53-mutant human tumor cell lines. In the human dedifferentiated liposarcoma cell line (LS141) and the p53 wild-type HCT116 cells, Nutlin-3a induced downregulation of E2F1 and this effect appeared to be proteasome dependent. In contrast, in MPNST and HCTp53-/- cells, Nutlin-3a inhibited the binding of E2F1 to MDM2 and induced transcriptional activation of free E2F1 in the presence of Cis-induced DNA damage. Downregulation of E2F1 by small interfering RNA significantly decreased the level of apoptosis induced by Cis and Nutlin-3a treatment. Moreover, expression of a dominant-negative form of E2F1 rescued cells from apoptosis, whereas cells overexpressing wild-type E2F1 showed an increase in cell death. This correlated with the induction of the proapoptotic proteins p73alpha and Noxa, which are both regulated by E2F1. These results indicate that antagonism of MDM2 by Nutlin-3a in cells with mutant p53 enhances chemosensitivity in an E2F1-dependent manner. Nutlin-3a therefore may provide a therapeutic benefit in tumors with mutant p53 provided it is combined with chemotherapy.


Available from: Grazia Ambrosini, Jun 09, 2015
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    • "To date, several p53-independent functions of Nutlin-3 have also been reported, including disruption of the MDM2–p73 interaction (Lau et al, 2008), sensitising p53-deficient chemoresistant cells to chemotherapy-induced apoptosis via upregulation of TAp73 and E2F1 (Ambrosini et al, 2007; Peirce and Findley, 2009), as well as acting as a competitive Multi-Drug Resistance Protein 1 (MDR-1) inhibitor and reversing MDR-1-mediated drug resistance (Michaelis et al, 2009). Acquired multidrug resistance is a major challenge in the successful treatment of cancers and can occur by increased expression of ATP-binding cassette (ABC) transporters, such as MDR-1, a 170-kDa transmembrane efflux pump encoded by the MDR-1 (ABCB1) gene. "
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    ABSTRACT: Background: A frequent mechanism of acquired multidrug resistance in human cancers is overexpression of ATP-binding cassette transporters such as the Multi-Drug Resistance Protein 1 (MDR-1). Nutlin-3, an MDM2-p53 antagonist, has previously been reported to be a competitive MDR-1 inhibitor. Methods: This study assessed whether the structurally diverse MDM2-p53 antagonists, MI-63, NDD0005, and RG7388 are also able to modulate MDR-1 function, particularly in p53 mutant neuroblastoma cells, using XTT-based cell viability assays, western blotting, and liquid chromatography-mass spectrometry analysis. Results: Verapamil and the MDM2-p53 antagonists potentiated vincristine-mediated growth inhibition in a concentration-dependent manner when used in combination with high MDR-1-expressing p53 mutant neuroblastoma cell lines at concentrations that did not affect the viability of cells when given alone. Liquid chromatography-mass spectrometry analyses showed that verapamil, Nutlin-3, MI-63 and NDD0005, but not RG7388, led to increased intracellular levels of vincristine in high MDR-1-expressing cell lines. Conclusions: These results show that in addition to Nutlin-3, other structurally unrelated MDM2-p53 antagonists can also act as MDR-1 inhibitors and reverse MDR-1-mediated multidrug resistance in neuroblastoma cell lines in a p53-independent manner. These findings are important for future clinical trial design with MDM2-p53 antagonists when used in combination with agents that are MDR-1 substrates.
    British Journal of Cancer 06/2014; 111(4). DOI:10.1038/bjc.2014.325 · 4.84 Impact Factor
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    • "It has been reported previously that p73 could be induced by a wide variety of chemotherapeutic drugs, including arsenic trioxide [9]. P73, which shares sequence homology with p53, is rarely mutated in human cancer, but has been reported to be inactivated by mutp53 or MDM2 [19,20]. We found the same phenomenon that p73 could be induced by arsenic trioxide in HCC cells here. "
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    ABSTRACT: Background Arsenic trioxide has been demonstrated as an effective anti-cancer drug against leukemia and solid tumors both in vitro and in vivo. However, recent phase II trials demonstrated that single agent arsenic trioxide was poorly effective against hepatocellular carcinoma (HCC), which might be due to drug resistance. Methods Mutation detection of p53 gene in arsenic trioxide resistant HCC cell lines was performed. The therapeutic effects of arsenic trioxide and Nutlin-3 on HCC were evaluated both in vitro and in vivo. A series of experiments including MTT, apoptosis assays, co-Immunoprecipitation, siRNA transfection, lentiviral infection, cell migration, invasion, and epithelial-mesenchy-mal transition (EMT) assays were performed to investigate the underlying mechanisms. Results The acquisition of p53 mutation contributed to arsenic trioxide resistance and enhanced metastatic potential of HCC cells. Mutant p53 (Mutp53) silence could re-sensitize HCC resistant cells to arsenic trioxide and inhibit the metastatic activities, while mutp53 overexpression showed the opposite effects. Neither arsenic trioxide nor Nutlin-3 could exhibit obvious effects against arsenic trioxide resistant HCC cells, while combination of them showed significant effects. Nutlin-3 can not only increase the intracellular arsenicals through inhibition of p-gp but also promote the p73 activation and mutp53 degradation mediated by arsenic trioxide. In vivo experiments indicated that Nutlin-3 can potentiate the antitumor activities of arsenic trioxide in an orthotopic hepatic tumor model and inhibit the metastasis to lung. Conclusions Acquisitions of p53 mutations contributed to the resistance of HCC to arsenic trioxide. Nutlin-3 could overcome arsenic trioxide resistance and inhibit tumor metastasis through p73 activation and promoting mutant p53 degradation mediated by arsenic trioxide.
    Molecular Cancer 05/2014; 13(1):133. DOI:10.1186/1476-4598-13-133 · 4.26 Impact Factor
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    • "In general, protein lysine acetylation has been shown to play an important role in regulation of cellular function and cancer cell signaling, also in AML [46,47]. In addition to inhibiting MDM2-p53 interaction and modulating p53, nutlin-3 may affect several other proteins, either as a consequence of p53 transcription-dependent or -independent effects [48], changed interactions between MDM2 and other proteins than p53 [11,28], or direct effect of nutlin-3 interaction with other proteins than MDM2 [12]. Accordingly, we wanted to examine if nutlin-3 could enhance the acetylation of other proteins than p53. "
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    ABSTRACT: Background The small-molecule MDM2 antagonist nutlin-3 has proved to be an effective p53 activating therapeutic compound in several preclinical cancer models, including acute myeloid leukemia (AML). We and others have previously reported a vigorous acetylation of the p53 protein by nutlin-treatment. In this study we aimed to investigate the functional role of this p53 acetylation in nutlin-sensitivity, and further to explore if nutlin-induced protein acetylation in general could indicate novel targets for the enhancement of nutlin-based therapy. Results Nutlin-3 was found to enhance the acetylation of p53 in the human AML cell line MOLM-13 (wild type TP53) and in TP53 null cells transfected with wild type p53 cDNA. Stable isotope labeling with amino acids in cell culture (SILAC) in combination with immunoprecipitation using an anti-acetyl-lysine antibody and mass spectrometry analysis identified increased levels of acetylated Histone H2B, Hsp27 and Hsp90 in MOLM-13 cells after nutlin-treatment, accompanied by downregulation of total levels of Hsp27 and Hsp90. Intracellular levels of heat shock proteins Hsp27, Hsp40, Hsp60, Hsp70 and Hsp90α were correlated to nutlin-sensitivity for primary AML cells (n = 40), and AML patient samples with low sensitivity to nutlin-3 tended to express higher levels of heat shock proteins than more responsive samples. Combination therapy of nutlin-3 and Hsp90 inhibitor geldanamycin demonstrated synergistic induction of apoptosis in AML cell lines and primary AML cells. Finally, TP53 null cells transfected with a p53 acetylation defective mutant demonstrated decreased heat shock protein acetylation and sensitivity to nutlin-3 compared to wild type p53 expressing cells. Conclusions Altogether, our results demonstrate that nutlin-3 induces acetylation of p53, histones and heat shock proteins, and indicate that p53 acetylation status and the levels of heat shock proteins may participate in modulation of nutlin-3 sensitivity in AML.
    Molecular Cancer 05/2014; 13(1):116. DOI:10.1186/1476-4598-13-116 · 4.26 Impact Factor
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