Nutlin

Manufactured by Merck Group

Sourced in Germany

Nutlin is a small-molecule inhibitor developed by Merck Group. It functions by blocking the interaction between the p53 tumor suppressor protein and the MDM2 (murine double minute 2) protein, which regulates p53 stability and activity.

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Lab products found in correlation

Close spelling variants of this product

Nutlin-3 (104 citations) Nutlin-3a (90 citations)

14 protocols using nutlin

Lipofectamine Transfection of ASOs

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Cultured cells were seeded out one day prior to transfection to allow the cells to adhere. After 24 h the cells were washed once in Opti-MEM (Gibco) and transfected using a mixture of Lipofectamine 2000 (5 µg/mL (Sk-Hep-1), 10 µg/mL (Hep3B), 10 µg/mL (HEK293) 10 µg/mL (THLE-3) ThermoFisher), Opti-MEM and the ASOs at a final concentration of 1, 5 or 25 nM. Four hours after transfection, the mixture was carefully removed from the cells and fresh medium was added to each well. HepG2 cells were reverse-transfected. A mixture of Lipofectamine 2000 (2.5 µg/mL, ThermoFisher), Opti-MEM combined with the ASOs at a final concentration of 1, 5 or 25 nM were incubated at RT, and the HepG2 cells were added after 15 min of incubation. Knockdown was assessed 48 h after transfection. The cells were harvested, total RNA isolated and RNA expression measured using RT-qPCR as described.
In the experiments in which the effect of PURPL knockdown was assesses in combination with Doxorubicin or Nutlin treatment, cells were transfected as described above with ASOs at a final concentration of 25 nM for 24 h before DMSO (0.5%), Doxorubicin (Sigma; 5 µM) or Nutlin (Sigma; 5 µM) was added to the appropriate wells. Both Doxorubin and Nutlin were reconstituted in DMSO (Sigma).

Berhane T., Holm A., Karstensen K.T., Petri A., Ilieva M.S., Krarup H., Vyberg M., Løvendorf M.B, & Kauppinen S. (2022). Knockdown of the long noncoding RNA PURPL induces apoptosis and sensitizes liver cancer cells to doxorubicin. Scientific Reports, 12, 19502.

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Dissolution of Nutlin, Etoposide, and Cisplatin

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Nutlin (Sigma) and etoposide (Sigma) were dissolved in DMSO (Sigma) at 17.2 mM and 50 mM, respectively, from which working stocks were generated. The concentration of Nutlin used refers to the entire mixture, but only half of the total concentration represents the active enantiomer A. Cisplatin (Sigma) was resuspended in 0.9% NaCl at 5 mM from which working stocks were generated.

Carrillo A.M., Hicks M., Khabele D, & Eischen C.M. (2015). Pharmacologically increasing Mdm2 inhibits DNA repair and cooperates with genotoxic agents to kill p53 inactivated ovarian cancer cells. Molecular cancer research : MCR, 13(8), 1197-1205.

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Nutlin and BHB Effects on U2OS Cell Proliferation

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U2OS cells were treated either with 10 µM Nutlin (Sigma) for 8 h, 10 mM BHB for 16 h, or 10 µM Nutlin for 8 h and 10 mM BHB for 16 h. Or they were treated either with 10 µM doxorubicin for 8 h, or with 10 mM BHB for 16 h, or with 10 µM doxorubicin for 8 h and 10 mM of BHB for 16 h.
The treated cells were then grown in the presence of 50 µM EdU for 2 h, fixed and permeabilized. EdU was used to visualize proliferating cells with a Click-iT® EdU Apollo Fluor® 567 Imaging kit. The cells were then counterstained with DAPI (4',6-diamidio-2-phenylindole) and imaged on a confocal microscope. In all, 20 randomly selected fields were imaged. The number of EdU-positive and DAPI-stained cells were counted and the percentage of cells with EdU-positive labeling was calculated. The experiments were repeated three times. All results are expressed as the mean ± standard deviation. Analysis of variance was performed using Student’s t test to determine the statistical significance of differences among the groups.

Liu K., Li F., Sun Q., Lin N., Han H., You K., Tian F., Mao Z., Li T., Tong T., Geng M., Zhao Y., Gu W, & Zhao W. (2019). p53 β-hydroxybutyrylation attenuates p53 activity. Cell Death & Disease, 10(3), 243.

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Apoptosis Induction Assay Protocol

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RITA was obtained from National Cancer Institute (NCI). Nutlin, 5-FU, ActD, CDDP, PFT-α, PFT-μ, cycloheximide, MG132, propidium iodide (PI) and Rhodamine 123 (Rho123) were from Sigma.

Li H., Zhang Y., Ströse A., Tedesco D., Gurova K, & Selivanova G. (2014). Integrated high-throughput analysis identifies Sp1 as a crucial determinant of p53-mediated apoptosis. Cell Death and Differentiation, 21(9), 1493-1502.

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Evaluating Lapatinib Effects on Breast Cancer

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Human Her2 positive breast cancer cell line BT474 (E285K p53 mutation) and human breast cancer cell line MCF7 (contain functional wtp53) were obtained from ATCC. Where indicated, cells were treated with indicated concentrations of lapatinib (LC lab, # L-4899). Concentrations of lapatinib were optimized for every experimental setting, depending on cell types. Where indicated cells were treated with 5 μM MG132 (Sigma) and 5 μM nutlin (Sigma) added to the medium. All cell viability assays were done using standard clonogenicity assays and CellTiter-Blue Cell Viability Assay (Promega, 96-well format with 5,000 cells/well seeded 24 hrs prior). Cells were treated for 48 hours in various concentrations of drug used. Florescence was detected by SPECTRAmax M2 (Molecular Devices).

Li D, & Marchenko N.D. (2016). ErbB2 inhibition by lapatinib promotes degradation of mutant p53 protein in cancer cells. Oncotarget, 8(4), 5823-5833.

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Tumor Organoid Establishment from Mouse Models

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Firrm^−/−;Trp53^−/− and Trp53^−/− organoids were derived from cryopreserved mammary tumor pieces and processed as previously described by Duarte et al. (47 (link)). Briefly, tumor material was dissected and digested using collagenase A (2 mg/ml; Gibco) in advanced DMEM/F12, washed in growth medium (advanced DMEM/F12, 10 mM Hepes, GlutaMAX, and penicillin-streptomycin) and filtered through a cell strainer. Organoids were then cultured in growth medium further supplemented with B27 (Gibco), 125 μM N-acetyl-l-cysteine (Sigma-Aldrich), and murine epidermal growth factor (50 ng/ml; Sigma-Aldrich). Cells were embedded in 1:1 Culturex Reduced Growth Factor Basement Membrane Extract (BME) Type 2 (Trevigen) mixed with medium and maintained at 37°C and 5% CO₂. To establish organoid cultures, cells were supplemented with 5 μM nutlin (Sigma-Aldrich) for the initial 3 weeks of culture.

Mazouzi A., Moser S.C., Abascal F., van den Broek B., Del Castillo Velasco-Herrera M., van der Heijden I., Hekkelman M., Drenth A.P., van der Burg E., Kroese L.J., Jalink K., Adams D.J., Jonkers J, & Brummelkamp T.R. (2023). FIRRM/C1orf112 mediates resolution of homologous recombination intermediates in response to DNA interstrand crosslinks. Science Advances, 9(22), eadf4409.

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Cytotoxicity Evaluation of Chemotherapeutics

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HU (Sigma-Aldrich, H8627-5G), CPT (Sigma-Aldrich, C9911−100MG), cisplatin (Merck KGaA, Darmstadt, Germany,60778-25EA), doxorubicin (Sigma-Aldrich, D1515−10MG), nutlin (Sigma-Aldrich, N6287−1MG) were applied as indicated. dNTPs analogs CldU (Sigma-Aldrich, C6891−100MG), IdU (Sigma-Aldrich, I7125-5G), and thymidine (Sigma-Aldrich,89270-5 G) were applied as indicated.

Statello L., Fernandez-Justel J.M., González J., Montes M., Ranieri A., Goñi E., Mas A.M, & Huarte M. (2024). The chromatin-associated lncREST ensures effective replication stress response by promoting the assembly of fork signaling factors. Nature Communications, 15, 978.

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Cell Line Maintenance and Nutlin Treatment

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HCT116, SJSA1 were maintained in RPMI (Corning); U2OS cells were maintained in DMEM (Corning). Media were supplemented with 10% FBS, antibiotics (100 units/mL penicillin plus 100 mg/mL streptomycin) and 2mM L-glutamine. SJSA1 p53KO cells were provided by the Espinosa lab. All cell lines were tested for mycoplasma contamination and authenticated by STR profiling (BMR Genomics). When needed cells at 70%–80% of confluence were treated with 10μM Nutlin (Sigma-Aldrich) dissolved in DMSO. The Nutlin-family MDM2 inhibitor RG7112 (Cayman Chemical) dissolved in DMSO was used for some experiments in SJSA1 cells at 5μM concentration based on published results (Tovar et al., 2013 (link)).

Rizzotto D., Zaccara S., Rossi A., Galbraith M.D., Andrysik Z., Pandey A., Sullivan K.D., Quattrone A., Espinosa J.M., Dassi E, & Inga A. (2020). Nutlin-Induced Apoptosis Is Specified by a Translation Program Regulated by PCBP2 and DHX30. Cell reports, 30(13), 4355-4369.e6.

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Evaluating Cisplatin Cytotoxicity in Cancer Cell Lines

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SJSA1, MHM, U2OS, MG63, SAOS, and MCF7 cells were obtained from ATCC. SJSA1 and MHM cells were grown in RPMI medium, MCF7, U2OS and MG63 in DMEM medium, and SAOS cells in McCoy's 5A medium with 10% fetal bovine serum (FBS), penicillin (100 U/mL) and streptomycin (100 μg/mL). Cells were plated 48h before being treated with Cisplatin (Bedford Laboratory) at the indicated concentrations. Nutlin, 2-D-glucose (2-DG), acridine orange (AO), monodansylcadaverine (MDC), bafilomycin A1, and chloroquine were obtained from Sigma Chemical Co (St. Louis, MO). MK2206 and LY2603618 were obtained from Selleck Chemicals. Oligomycin, glucose and 2-DG were prepared following manufacturer's instructions that were supplied in the XF glycolysis test kit (Seahorse Bioscience, Billerica, MA). GFP-LC3 plasmid was previously described [47 (link)]. pMXs-puro GFP-p62 (Plasmid #38277) was obtained from Addgene. Dihydroethidium (DHE) were purchased from Invitrogen.

Duan L., Perez R.E., Davaadelger B., Dedkova E.N., Blatter L.A, & Maki C.G. (2015). p53-regulated autophagy is controlled by glycolysis and determines cell fate. Oncotarget, 6(27), 23135-23156.

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Cell Treatment and Harvest Protocol

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Human lymphoblastoid cells were seeded at 5 × 10⁶ cells per experimental condition, MEFs, Yumm1.7 cells, M93-047 cells, and HCT116 cells were seeded at 1 × 10⁶ to 2 × 10⁶ cells per experimental condition unless otherwise noted. Cells were treated with 10 μmol/L nutlin (Sigma), 5 to 10 μmol/L cisplatin (MedChemExpress), 5 to 10 μmol/L etoposide (SelleckChem), 25 μg/mL cycloheximide (Sigma), 1 μmol/L or 0.1 μmol/L doxorubicin (Cayman), 40 μmol/L CB-839 (SelleckChem), or the appropriate vehicle control for the indicated time points. Cells were harvested at indicated time points and collected by centrifugation at 2,000 rpm for 5 minutes at 4°C, followed by a cold PBS (Corning) wash and additional centrifugation.

Indeglia A., Leung J.C., Miller S.A., Leu J.I., Dougherty J.F., Clarke N.L., Kirven N.A., Shao C., Ke L., Lovell S., Barnoud T., Lu D.Y., Lin C., Kannan T., Battaile K.P., Yang T.H., Batista Oliva I., Claiborne D.T., Vogel P., Liu L., Liu Q., Nefedova Y., Cassel J., Auslander N., Kossenkov A.V., Karanicolas J, & Murphy M.E. (2023). An African-Specific Variant of TP53 Reveals PADI4 as a Regulator of p53-Mediated Tumor Suppression. Cancer Discovery, 13(7), 1696-1719.

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