nutlin-3a and Neoplasms

Disruption of the MDM2-p53 protein-protein interaction by small-molecule inhibitors has been highly pursued by many academic laboratories and pharmaceutical companies as a promising strategy for cancer therapy. To date, based on the explanation of the cocrystal structure of MDM2 with p53, many highly potent and selective small-molecule MDM2 inhibitors have been successfully discovered and nine of them are currently under different clinical trials for cancer therapy. Herein, we will review the function of MDM2 and provide a comprehensive and updated overview of small-molecule MDM2 inhibitors in different clinical phases for cancer therapy, especially focusing on the identification and optimization, and preclinical/clinical studies of these clinical-stage MDM2 inhibitors. Challenges regarding acquired resistance and potential toxicity of MDM2 inhibitors to normal tissues and outlook are also briefly discussed, which will further guide the design of new small-molecule MDM2 inhibitors.

Topics: Antineoplastic Agents; Humans; Neoplasms; Protein Binding; Proto-Oncogene Proteins c-mdm2; Tumor Suppressor Protein p53

Design of small-molecule inhibitors (MDM2 inhibitors) to block the MDM2-p53 protein-protein interaction has been pursued as a new cancer therapeutic strategy. In recent years, potent, selective, and efficacious MDM2 inhibitors have been successfully obtained and seven such compounds have been advanced into early phase clinical trials for the treatment of human cancers. Here, we review the design, synthesis, properties, preclinical, and clinical studies of these clinical-stage MDM2 inhibitors.

Topics: Animals; Clinical Trials as Topic; Enzyme Inhibitors; Humans; Models, Molecular; Molecular Structure; Neoplasms; Protein Binding; Proto-Oncogene Proteins c-mdm2; Small Molecule Libraries; Structure-Activity Relationship; Tumor Suppressor Protein p53

A peculiar feature of several types of childhood cancer is that loss-of-function mutations of the TP53 (p53) tumor suppressor gene are uncommon, in contrast to many adult tumors. As p53 needs to be inactivated in order for tumor cells to survive and thrive, pediatric tumors typically make use of other mechanisms to keep p53 in check. One of the critical negative regulators of p53 is the MDM2 oncoprotein. Many anticancer drug development efforts in the past decade have therefore been devoted to the discovery and optimization of small molecules that selectively disrupt the interaction between MDM2 and p53, which could provide, in principle, a potent means to restore p53 function in tumor cells with wild-type p53. The nutlins are the class of selective inhibitors of the p53-MDM2 interaction that are currently most advanced in their clinical development. We review here the preclinical data that support the potential therapeutic use of nutlin drugs in the treatment of various pediatric tumors, including neuroblastoma, retinoblastoma, osteosarcoma, Ewing's sarcoma, rhabdomyosarcoma, medulloblastoma, and childhood acute lymphoblastic leukemia.

Topics: Animals; Child; Genes, p53; Humans; Imidazoles; Neoplasms; Piperazines; Tumor Suppressor Protein p53

Since the discovery of p53 as "guardian of the genome", a large number of efforts have been put in place in order to find molecular strategies aiming to restore p53 wild-type functions, particularly in the light of the fact that its pathway results ineffective in most tumors even though they have non-mutated p53. In this context, pediatric cancers, that are mostly p53 wild-type at the time of diagnosis, represent an ideal target for such therapeutic approach. Within the several mechanisms and proteins ruling p53 activity, the murine double minute 2 (MDM2) is its crucial negative regulator, frequently found overexpressed in p53-wild-type tumors. The development of new technologies such as nuclear magnetic resonance structure analyses, computational structure-based design studies, and library peptides screening have recently led to the discovery and characterization of a large number of compounds belonging to different chemical families that are able to target the interaction p53-MDM2, rescuing the p53 wild-type pathway with an overall pro-apoptotic and anticancer activity. Within the preclinical assessment of these molecules, the cis-imidazoline analogue Nutlin-3 has definitely attracted great interest for its in vitro and in vivo antitumor activity in several pediatric cancer models, either as single agent on in combination with standard chemotherapy. In this light, the aim of this review is to summarize the main preclinical evidences of the potential of MDM2 inhibitors for the treatment of childhood cancers and the key suggestions coming from their assessment in the treatment of adult cancers as proof of concept for future pediatric clinical studies.

Topics: Child; Enzyme Inhibitors; Humans; Imidazoles; Neoplasms; Piperazines; Protein Binding; Protein Interaction Domains and Motifs; Proto-Oncogene Proteins c-mdm2; TNF-Related Apoptosis-Inducing Ligand; Tumor Suppressor Protein p53

For last few decades, the active site cleft and substrate-binding site of enzymes as well as ligand-binding site of the receptors have served as the main pharmacological space for drug discovery. However, rapid accumulation of proteome and protein network analysis data has opened a new therapeutic space that is the interface between the interacting proteins. Due to the complexity of the interaction modes and the numbers of the participating components, it is still challenging to identify the chemicals that can accurately control the protein-protein interactions at desire. Nonetheless, the number of chemical drugs and candidates working at the interface of the interacting proteins are rapidly increasing. This review addresses the current case studies and state-of-the-arts in the development of small chemical modulators controlling the interactions of the proteins that have pathological implications in various human diseases such as cancer, immune disorders, neurodegenerative and infectious diseases.

Topics: Animals; Anti-Bacterial Agents; Antineoplastic Agents; Antiviral Agents; Bacterial Infections; Drug Discovery; Humans; Immune System Diseases; Immunologic Factors; Models, Molecular; Neoplasms; Neurodegenerative Diseases; Protein Interaction Maps; Virus Diseases

Restoring p53 activity by inhibiting the interaction between p53 and MDM2 represents an attractive approach for cancer therapy. To this end, a number of small-molecule p53-MDM2 binding inhibitors have been developed during the past several years. Nutlin-3 is a potent and selective small-molecule MDM2 antagonist that has shown considerable promise in pre-clinical studies. This review will highlight recent advances in the development of small-molecule MDM2 antagonists as potential cancer therapeutics, with special emphasis on Nutlin-3.

Topics: Antineoplastic Agents; Genes, p53; Humans; Imidazoles; Molecular Targeted Therapy; Neoplasms; Piperazines; Protein Binding; Proto-Oncogene Proteins c-mdm2; Tumor Suppressor Protein p53

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.

Topics: Antineoplastic Agents; Antineoplastic Combined Chemotherapy Protocols; Drug Synergism; Genes, p53; Humans; Imidazoles; Neoplasms; Piperazines; Proto-Oncogene Proteins c-mdm2; Tumor Suppressor Protein p53

Radioresistance stands as a fundamental barrier that limits the effectiveness of radiotherapy in cancer treatment. Recent evidences suggest that radioresistance is due to tumour repopulation and involves several signalling pathways, including p53/MDM2 interaction. Ionizing radiation induces p53-dependent MDM2 gene transcription that, in turn, inhibits p53 transcriptional activity, favouring its nuclear export and stimulating its degradation. In light of the observation that in many human tumours the inadequate function of p53 is the result of MDM2 over-expression, several authors have considered as an attractive therapeutic strategy to activate p53 signalling in tumours by inhibiting MDM2 activities or p53/MDM2 interaction. We retain that, by preventing the interaction p53/MDM2 with Nutlin, a small molecule that bind at the interface between these two proteins, the effectiveness of ionizing radiation treatment could be improved. Promising results have recently emerged from in vitro studies performed on laryngeal, prostate and lung cancer cell lines treated with Nutlin in combination with ionizing radiation. Based on these findings, we believe that the combined approach Nutlin/ionizing radiation should be further investigated for efficacy on both solid tumours and lymphoproliferative disorders as well as for side effects on normal cells and tissues. Therefore, the purpose of this review is to report the first results obtained by using Nutlins alone or in combination with other therapeutic agents on primary tumour cells, in vitro cell lines or tumour xenografts and to present the most recent advances in the understanding of the molecular mechanisms underlining ionizing radiation cytotoxicity and resistance.

Topics: Animals; Antineoplastic Agents; Apoptosis; Cell Line, Tumor; Combined Modality Therapy; Humans; Imidazoles; Neoplasms; Piperazines; Proto-Oncogene Proteins c-mdm2; Radiation, Ionizing; Tumor Suppressor Protein p53

Tumor suppressor p53 is an attractive cancer therapeutic target because it can be functionally activated to eradicate tumors. Direct gene alterations in p53 or interaction between p53 and MDM2 proteins are two alternative mechanisms for the inactivation of p53 function. Designing small molecules to block the MDM2-p53 interaction and reactivate the p53 function is a promising therapeutic strategy for the treatment of cancers retaining wild-type p53. This review will highlight recent advances in the design and development of small-molecule inhibitors of the MDM2-p53 interaction as new cancer therapies. A number of these small-molecule inhibitors, such as analogs of MI-219 and Nutlin-3, have progressed to advanced preclinical development or early phase clinical trials.

Topics: Animals; Antineoplastic Agents; Cell Cycle Proteins; Clinical Trials as Topic; Drug Design; Drug Evaluation, Preclinical; Humans; Imidazoles; Neoplasms; Nuclear Proteins; Piperazines; Proto-Oncogene Proteins; Proto-Oncogene Proteins c-mdm2; Tumor Suppressor Protein p53

The tumor suppressor p53 is a powerful antitumoral molecule frequently inactivated by mutations or deletions in cancer. However, half of all human tumors express wild-type p53, and its activation by antagonizing its negative regulator murine double minute 2 (MDM2) might offer a new therapeutic strategy. Proof-of-concept experiments have demonstrated the feasibility of this approach in vitro but the development of pharmacological inhibitors has been challenging. Recently, potent and selective small-molecule MDM2 inhibitors have been identified. Studies with these compounds have strengthened the concept that selective, non-genotoxic p53 activation is a viable alternative to current cytotoxic chemotherapy but clinical validation is still pending. Here, the new developments in the quest for pharmacological p53 activators are reviewed with an emphasis on small-molecule inhibitors of the p53-MDM2 interaction.

Topics: Benzodiazepines; Gene Expression Regulation, Neoplastic; Humans; Imidazoles; Models, Biological; Models, Molecular; Molecular Structure; Neoplasms; Oligonucleotides, Antisense; Piperazines; Protein Binding; Proto-Oncogene Proteins c-mdm2; Spiro Compounds; Tumor Suppressor Protein p53; Ubiquitin-Protein Ligases

Topics: Angiogenesis Inhibitors; Animals; Feedback, Physiological; Homeostasis; Imidazoles; Neoplasms; Neovascularization, Pathologic; Piperazines; Proto-Oncogene Proteins c-mdm2; Tumor Suppressor Protein p53

Topics: Animals; Anticarcinogenic Agents; Antineoplastic Agents; Carrier Proteins; Humans; Imidazoles; Models, Molecular; Neoplasms; Piperazines; Protein Binding; Tumor Suppressor Protein p53

Activation of p53 by small molecule MDM2 inhibitors can induce cell cycle arrest or death in p53 wildtype cancer cells. However, cancer cells exposed to hypoxia can develop resistance to other small molecules, such as chemotherapies, that activate p53. Here, we evaluated whether hypoxia could render cancer cells insensitive to two MDM2 inhibitors with different potencies, nutlin-3a and navtemadlin. Inhibitor efficacy and potency were evaluated under short-term hypoxic conditions in human and mouse cancer cells expressing different p53 genotypes (wild-type, mutant, or null). Treatment of wild-type p53 cancer cells with MDM2 inhibitors reduced cell growth by > 75% in hypoxia through activation of the p53-p21 signaling pathway; no inhibitor-induced growth reduction was observed in hypoxic mutant or null p53 cells except at very high concentrations. The concentration of inhibitors needed to induce the maximal p53 response was not significantly different in hypoxia compared to normoxia. However, inhibitor efficacy varied by species and by cell line, with stronger effects at lower concentrations observed in human cell lines than in mouse cell lines grown as 2D and 3D cultures. Together, these results indicate that MDM2 inhibitors retain efficacy in hypoxia, suggesting they could be useful for targeting acutely hypoxic cancer cells.

Topics: Animals; Antineoplastic Agents; Apoptosis; Cell Line, Tumor; Humans; Hypoxia; Mice; Neoplasms; Proto-Oncogene Proteins c-mdm2; Tumor Suppressor Protein p53

Dysfunction of p53 and resistance to cancer drugs can arise through mutually exclusive overexpression of MDM2 or MDM4. Cisplatin-resistant cells, however, can demonstrate increased binding of both MDM2 and MDM4 to p53 but in absence of cellular overexpression. Whether MDM2 inhibitors alone can activate p53 in these resistant cells was investigated with the goal to establish the mechanism for potential synergy with cisplatin. Thus, growth inhibition by individual drugs and combinations was assessed by a colorimetric assay. Drug-treated parental A2780 and resistant tumor cells were also examined for protein expression using immunoblot and reverse phase protein array (RPPA) and then subjected to Ingenuity Pathway Analysis (IPA). Gene expression was assessed by real-time polymerase chain reaction, DNA damage by confocal microscopy, cell cycle by flow cytometry, and homologous recombination (HR) by a GFP reporter assay. Our results demonstrate that Nutlin-3 but not RITA (reactivation of p53 and induction of tumor cell apoptosis) effectively disrupted the p53-MDM2-MDM4 complex to activate p53, which increased robustly with cisplatin/Nutlin-3 combination and enhanced antitumor effects more than either agent alone. RPPA, IPA, and confocal microscopy provided evidence for an "apparent" increase in DNA damage resulting from HR inhibition by cisplatin/Nutlin-3. Molecularly, the specific HR protein Rad51 was severely downregulated by the combination via two mechanisms: p53-dependent transrepression and p53/MDM2-mediated proteasomal degradation. In conclusion, Nutlin-3 fully destabilizes the p53-MDM2-MDM4 complex and synergizes with cisplatin to intensify p53 function, which then downregulates Rad51 through a bimodal mechanism. As a result, HR is inhibited and antitumor activity enhanced in otherwise HR-proficient sensitive and resistant tumor cells. SIGNIFICANCE STATEMENT: Rad51 downregulation by the combination of cisplatin and Nutlin-3 inhibits homologous recombination (HR), which leads to persistence in DNA damage but not an increase. Thus, inhibition of HR enhances antitumor activity in otherwise HR-proficient sensitive and resistant tumor cells.

Topics: Antineoplastic Combined Chemotherapy Protocols; Cell Cycle Proteins; Cell Line, Tumor; Cell Proliferation; Cisplatin; DNA Damage; Down-Regulation; Drug Resistance, Neoplasm; Drug Synergism; Furans; Gene Expression Regulation, Neoplastic; Homologous Recombination; Humans; Imidazoles; Neoplasms; Piperazines; Proto-Oncogene Proteins; Proto-Oncogene Proteins c-mdm2; Rad51 Recombinase; Tumor Suppressor Protein p53

Data from several large high-throughput drug response screens have become available to the scientific community recently. Although many efforts have been made to use this information to predict drug sensitivity, our ability to accurately predict drug response based on genetic data remains limited. In order to systematically examine how different aspects of modelling affect the resulting prediction accuracy, we built a range of models for seven drugs (erlotinib, pacliatxel, lapatinib, PLX4720, sorafenib, nutlin-3 and nilotinib) using data from the largest available cell line and xenograft drug sensitivity screens. We found that the drug response metric, the choice of the molecular data type and the number of training samples have a substantial impact on prediction accuracy. We also compared the tasks of drug response prediction with tissue type prediction and found that, unlike for drug response, tissue type can be predicted with high accuracy. Furthermore, we assessed our ability to predict drug response in four xenograft cohorts (treated either with erlotinib, gemcitabine or paclitaxel) using models trained on cell line data. We could predict response in an erlotinib-treated cohort with a moderate accuracy (correlation ≈ 0.5), but were unable to correctly predict responses in cohorts treated with gemcitabine or paclitaxel.

Topics: Animals; Biomarkers, Pharmacological; Cell Line, Tumor; Erlotinib Hydrochloride; Humans; Imidazoles; Indoles; Lapatinib; Machine Learning; Mice; Neoplasms; Organ Specificity; Paclitaxel; Piperazines; Prognosis; Pyrimidines; Sorafenib; Sulfonamides; Xenograft Model Antitumor Assays

Activated p53 can promote apoptosis or cell cycle arrest. Differences in energy metabolism can influence cell fate in response to activated p53. Nutlin-3a is a preclinical drug and small molecule activator of p53. Alpha-ketoglutarate (αKG) levels were reduced in cells sensitive to Nutlin-3a-induced apoptosis and increased in cells resistant to this apoptosis. Add-back of a cell-permeable αKG analog (DMKG) rescued cells from apoptosis in response to Nutlin-3a. OGDH is a component of the αKGDH complex that converts αKG to succinate. OGDH knockdown increased endogenous αKG levels and also rescued cells from Nutlin-3a-induced apoptosis. We previously showed reduced autophagy and ATG gene expression contributes to Nutlin-3a-induced apoptosis. DMKG and OGDH knockdown restored autophagy and ATG gene expression in Nutlin-3a-treated cells. These studies indicate αKG levels, regulated by p53 and OGDH, determine autophagy and apoptosis in response to Nutlin-3a.

Topics: A549 Cells; Apoptosis; Autophagy; Bone Neoplasms; Cell Line, Tumor; Gene Knockdown Techniques; Glycolysis; Humans; Imidazoles; Ketoglutarate Dehydrogenase Complex; Ketoglutaric Acids; Lung Neoplasms; Mechanistic Target of Rapamycin Complex 1; Neoplasms; Osteosarcoma; Piperazines; Tumor Suppressor Protein p53

Activation of p53 may induce apoptosis or cellular senescence in stressed cells. We here report that epidermal growth factor receptor (EGFR) is downregulated by p53 activation in a subset of cancer cell lines, and this EGFR downregulation mediates cellular senescence caused by p53 activation. EGFR confers resistance to senescence by sustaining the ERK signaling. DYRK1A (dual-specificity tyrosine-phosphorylated and tyrosine-regulated kinase 1A), an EGFR-stabilizing kinase, is downregulated by p53 and, when ectopically expressed, can attenuate p53 activation-induced EGFR reduction and cellular senescence. We further showed that the increased degradation of DYRK1A caused by p53 activation was mediated by MDM2. MDM2 was found to physically interact with and ubiquitinate DYRK1A, ultimately leading to its proteosomal degradation. Importantly, administration of Nutlin-3a, which disrupts the binding of MDM2 to p53, but not that of MDM2 to DYRK1A, reduced the levels of DYRK1A and EGFR, induced senescence, and inhibited growth of tumor xenografts formed by U87 glioblastoma cells. Ectopic expression of EGFR in tumor xenografts attenuated senescence and tumor reduction caused by Nultin-3a. Our findings thus established a novel link between p53 and EGFR and may have implications in p53 activation-based therapies.

Topics: A549 Cells; Animals; Cellular Senescence; Dyrk Kinases; ErbB Receptors; HCT116 Cells; Heterografts; Humans; Imidazoles; MCF-7 Cells; Mice; Mice, Inbred BALB C; Mice, Nude; Neoplasms; Piperazines; Protein Serine-Threonine Kinases; Protein-Tyrosine Kinases; Proto-Oncogene Proteins c-mdm2; Transfection; Tumor Burden; Tumor Suppressor Protein p53

Prenylation of several bioactive scaffolds is a very interesting strategy used in Medicinal Chemistry in order to improve biological/pharmacological effects. A small library of prenylchalcones was synthesized and evaluated for the ability to inhibit the MDM2-p53 interaction using a yeast-based assay. The capacity of all synthesized prenylchalcones and their non-prenylated precursors to inhibit the growth of human colon tumor HCT116 cells was also evaluated. The obtained results led to the identification of a hit compound, prenylchalcone 2e, which behaved as potential inhibitor of the MDM2-p53 interaction in yeast, and showed improved cytotoxicity against human tumor cells expressing wild-type p53, including liver hepatocellular carcinoma HepG2, breast adenocarcinoma MCF-7, and malignant melanoma A375 cells. In colon cancer cells, it was also shown that the growth inhibitory effect of prenylchalcone 2e was associated with the induction of cell cycle arrest, apoptosis, and increased protein expression levels of p53 transcriptional targets. Moreover, computational docking studies were performed in order to predict docking poses and residues involved in the MDM2-p53 potential interaction.

Topics: Antineoplastic Agents; Apoptosis; Cell Cycle Checkpoints; Cell Line, Tumor; Cell Proliferation; Chalcone; HCT116 Cells; Humans; Models, Molecular; Molecular Docking Simulation; Neoplasms; Protein Interaction Maps; Proto-Oncogene Proteins c-mdm2; Small Molecule Libraries; Tumor Suppressor Protein p53

Nutlin-3a is a MDM2 antagonist and preclinical drug that activates p53. Cells with MDM2 gene amplification are especially prone to Nutlin-3a-induced apoptosis, though the basis for this is unclear. Glucose metabolism can inhibit apoptosis in response to Nutlin-3a through mechanisms that are incompletely understood. Glucose metabolism through the pentose phosphate pathway (PPP) produces NADPH that can protect cells from potentially lethal reactive oxygen species (ROS). We compared apoptosis and glucose metabolism in cancer cells with and without MDM2 gene amplification treated with Nutlin-3a. Apoptosis in MDM2-amplified cells was associated with a reduction in glycolysis and the PPP, reduced NADPH, increased ROS, and depletion of the transcription factor SP1, which normally promotes PPP gene expression. In contrast, glycolysis and the PPP were maintained or increased in MDM2 non-amplified cells treated with Nutlin-3a. This was dependent on p53-mediated AKT activation and was associated with maintenance of SP1 and continued expression of PPP genes. Knockdown or inhibition of AKT, SP1, or the PPP sensitized MDM2-non-amplified cells to apoptosis. The data indicate that p53 promotes AKT and SP1-dependent activation of the PPP that protects cells from Nutlin-3a-induced apoptosis. These findings provide insight into how glucose metabolism reduces Nutlin-3a-induced apoptosis, and also provide a mechanism for the heightened sensitivity of MDM2-amplified cells to apoptosis in response to Nutlin-3a.

Topics: Antineoplastic Agents; Apoptosis; Cell Line, Tumor; Glycolysis; Humans; Imidazoles; Neoplasms; Pentose Phosphate Pathway; Piperazines; Proto-Oncogene Proteins c-akt; Proto-Oncogene Proteins c-mdm2; Sp1 Transcription Factor; Tumor Suppressor Protein p53

The tumor suppressor protein p53 is central to the cellular stress response and may be a predictive biomarker for cancer treatments. Upon stress, wildtype p53 accumulates in the nucleus where it enforces cellular responses, including cell cycle arrest and cell death. p53 is so dominant in its effects, that p53 enforcement - or - restoration therapy is being studied for anti-cancer therapy. Two mechanistically distinct small molecules that act via p53 are the selective inhibitor of nuclear export, selinexor, and MDM2 inhibitor, nutlin-3a. Here, individual cells are studied to define cell cycle response signatures, which captures the variability of responses and includes the impact of loss of p53 expression on cell fates. The individual responses are then used to build the population level response. Matched cell lines with and without p53 expression indicate that while loss-of-function results in altered cell cycle signatures to selinexor treatment, it does not diminish overall cell loss. On the contrary, response to single-agent nutlin-3a shows a strong p53-dependence. Upon treatment with both selinexor and nutlin-3a there are combination effects in at least some cell lines - even when p53 is absent. Collectively, the findings indicate that p53 does act downstream of selinexor and nutlin-3a, and that p53 expression is dispensable for selinexor to cause cell death, but nutlin-3a response is more p53-dependent. Thus, TP53 disruption and lack of expression may not predict poor cell response to selinexor, and selinexor's mechanism of action potentially provides for strong efficacy regardless of p53 function.

Topics: Antineoplastic Agents; Apoptosis; Cell Cycle; Cell Cycle Checkpoints; Cell Line, Tumor; Cell Lineage; Cell Survival; Exportin 1 Protein; G1 Phase; Humans; Hydrazines; Imidazoles; Karyopherins; Neoplasms; Piperazines; Proto-Oncogene Proteins c-mdm2; Receptors, Cytoplasmic and Nuclear; Triazoles; Tumor Suppressor Protein p53

Topics: Antineoplastic Agents; Apoptosis; Bortezomib; Calcium; Dilatation; Drug Synergism; Endoplasmic Reticulum; HCT116 Cells; HeLa Cells; Humans; Imidazoles; Mitochondria; Neoplasms; Piperazines; Proteasome Endopeptidase Complex; Proteasome Inhibitors; Transcription Factor CHOP; Tumor Suppressor Protein p53

p53-independent malignant cancer is still severe health problem of human beings. HIF-1 pathway is believed to play an important role in the survival and developing progress of such cancers. In the present study, with the aim to inhibit the proliferation of p53-independent malignant cells, we disclose the optimization of 6a, the starting compound which is discovered in the screening of in-house compound collection. The structure-activity relationship (SAR) is summarized. The most potent derivative 8d, inhibits the proliferation of both p53-null and p53-mutated cells through inhibition of HIF-1 pathway. Our findings here provide a new chemotype in designing potent anticancer agent especially against those p53-independent malignant tumors.

Topics: Antineoplastic Agents; Benzofurans; Cell Cycle Checkpoints; Cell Line, Tumor; Cell Proliferation; Dose-Response Relationship, Drug; Drug Discovery; Drug Screening Assays, Antitumor; HCT116 Cells; Humans; Hypoxia-Inducible Factor 1; MCF-7 Cells; Molecular Structure; Neoplasms; Structure-Activity Relationship; Tumor Suppressor Protein p53

Many p53 hotspot mutants not only lose the transcriptional activity, but also show dominant-negative (DN) and oncogenic gain-of-function (GOF) activities. Increasing evidence indicates that knockdown of mutant p53 (mutp53) in cancer cells reduces their aggressive properties, suggesting that survival and proliferation of cancer cells are, at least partially, dependent on the presence of mutp53. However, these p53 siRNAs can downregulate both wild-type p53 (wtp53) and mutp53, which limits their therapeutic applications. In order to specifically deplete mutp53, we have developed allele-specific siRNAs against p53 hotspot mutants and validated their biological effects in the absence or presence of wtp53. First, the mutp53-specific siRNAs selectively reduced protein levels of matched p53 mutants with minimal reduction in wtp53 levels. Second, downregulation of mutp53 in cancer cells expressing a mutp53 alone (p53mut) resulted in significantly decreased cell proliferation and migration. Third, transfection of mutp53-specific siRNAs in cancer cells expressing both wtp53 and mutp53 also reduced cell proliferation and migration with increased transcripts of p53 downstream target genes, which became further profound when cells were treated with an MDM2 inhibitor Nutlin-3a or a chemotherapeutic agent doxorubicin. These results indicate that depletion of mutp53 by its specific siRNA restored endogenous wtp53 activity in cells expressing both wtp53 and mutp53. This is the first study demonstrating biological effects and therapeutic potential of allele-specific silencing of mutp53 by mutp53-specific siRNAs in cancer cells expressing both wtp53 and mutp53, thus providing a novel strategy towards targeted cancer therapies.

Topics: Alleles; Animals; Apoptosis; Blotting, Western; Carcinogenesis; Cell Adhesion; Cell Movement; Cell Proliferation; Down-Regulation; Genes, Dominant; Humans; Imidazoles; Immunoenzyme Techniques; Mice; Mutant Proteins; Mutation; Neoplasms; Piperazines; Real-Time Polymerase Chain Reaction; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; RNA, Small Interfering; Tumor Cells, Cultured; Tumor Suppressor Protein p53

Sensitivity to MDM2 inhibitors is widely different among responsive TP53 wild-type cell lines and tumors. Understanding the determinants of MDM2 inhibitor sensitivity is pertinent for their optimal clinical application. Wild-type p53-inducible phosphatase-1 (WIP1) encoded by PPM1D, is activated, gained/amplified in a range of TP53 wild-type malignancies, and is involved in p53 stress response homeostasis. We investigated cellular growth/proliferation of TP53 wild-type and matched mutant/null cell line pairs, differing in PPM1D genetic status, in response to Nutlin-3/RG7388 ± a highly selective WIP1 inhibitor, GSK2830371. We also assessed the effects of GSK2830371 on MDM2 inhibitor-induced p53(Ser15) phosphorylation, p53-mediated global transcriptional activity, and apoptosis. The investigated cell line pairs were relatively insensitive to single-agent GSK2830371. However, a non-growth-inhibitory dose of GSK2830371 markedly potentiated the response to MDM2 inhibitors in TP53 wild-type cell lines, most notably in those harboring PPM1D-activating mutations or copy number gain (up to 5.8-fold decrease in GI50). Potentiation also correlated with significant increase in MDM2 inhibitor-induced cell death endpoints that were preceded by a marked increase in a WIP1 negatively regulated substrate, phosphorylated p53(Ser15), known to increase p53 transcriptional activity. Microarray-based gene expression analysis showed that the combination treatment increases the subset of early RG7388-induced p53 transcriptional target genes. These findings demonstrate that potent and selective WIP1 inhibition potentiates the response to MDM2 inhibitors in TP53 wild-type cells, particularly those with PPM1D activation or gain, while highlighting the mechanistic importance of p53(Ser15) and its potential use as a biomarker for response to this combination regimen.

Topics: Aminopyridines; Antineoplastic Agents; Apoptosis; Caspase 3; Caspase 7; Catalysis; Cell Line, Tumor; Cell Survival; Dipeptides; Dose-Response Relationship, Drug; Drug Resistance, Neoplasm; Drug Synergism; Humans; Imidazoles; Mutation; Neoplasms; para-Aminobenzoates; Piperazines; Protein Phosphatase 2C; Proteolysis; Proto-Oncogene Proteins c-mdm2; Pyrrolidines; Transcription, Genetic; Tumor Suppressor Protein p53; Ubiquitin

The MDM2-p53 pathway has a prominent oncogenic function in the pathogenesis of various cancers. Nutlin-3, a small-molecule antagonist of MDM2-p53 interaction, inhibits proliferation in cancer cells with wild-type p53. Herein, we evaluate the expression of MDM2, both the full length and a splicing variant MDM2-A, and the sensitivity of Nutlin-3 in different cancer cell lines. Included are seven cell lines with wild-type p53 (four mesothelioma, one breast cancer, one chondrosarcoma, and one leiomyosarcoma), two liposarcoma cell lines harboring MDM2 amplification and wild-type p53, and one mesothelioma cell line harboring a p53 point mutation. Nutlin-3 treatment increased expression of cyclin D1, MDM2, and p53 in cell lines with wild-type p53. Additive effects were observed in cells containing wild-type p53 through coordinated attack on MDM2-p53 binding and cyclin D1 by lentivirual shRNA knockdown or small molecule inhibition, as demonstrated by immunoblots and cell viability analyses. Further results demonstrate that MDM2 binds to cyclin D1, and that an increase in cyclin D1 expression after Nutlin-3 treatment is correlated with expression and ubiquitin E3-ligase activity of MDM2. MDM2 and p53 knockdown experiments demonstrated inhibition of cyclin D1 by MDM2 but not p53. These results indicate that combination inhibition of cyclin D1 and MDM2-p53 binding warrants clinical evaluation as a novel therapeutic strategy in cancer cells harboring wild-type p53.

Topics: Cell Line, Tumor; Cyclin D1; Down-Regulation; Gene Knockdown Techniques; HEK293 Cells; Humans; Imidazoles; MCF-7 Cells; Neoplasms; Piperazines; Proto-Oncogene Proteins c-mdm2; Tumor Suppressor Protein p53

Treating cancer is one of the big challenges of this century and it has become evident that single chemotherapeutic treatment is rarely effective. As tumors often carry multiple mutations using combination therapy which addresses different targets seems therefore more beneficial. One of the most frequently mutated genes in tumors is the tumor suppressor p53. Significant work has been put in the development of p53 activators, which are now in clinical studies against diverse cancers. Recently, we could show that inhibition of V-ATPase, a multisubunit proton pump, by archazolid induces p53 protein levels in cancer cells. In this study, we provide evidence that the combination of archazolid with the p53 activator nutlin-3a is synergistically inducing cell death in different p53 wild type tumor cell lines. Mechanistically, this effect could presumably be attributed to reduction of glycolysis as TIGAR mRNA levels were increased and glucose uptake and Glut1 protein levels were reduced. In addition, combination treatment highly activated pro-apoptotic pathways including IGFBP3 and Bax inducing caspase-9 and PARP cleavage. Remarkably, combination of archazolid and nutlin-3a was more efficient in reducing tumor growth compared to single dose treatment in a U87MG mouse model in vivo. Hence, our findings suggest the combination of archazolid and nutlin-3a as a highly promising strategy for the treatment of p53 wild type tumors.

Topics: Adenosine Triphosphatases; Animals; Apoptosis; bcl-2-Associated X Protein; Cell Death; Cell Line, Tumor; Cell Nucleus; Cell Proliferation; Female; Glycolysis; Humans; Imidazoles; Insulin-Like Growth Factor Binding Protein 3; Mice, Inbred BALB C; Mice, SCID; Neoplasms; Piperazines; Proto-Oncogene Proteins c-mdm2; Signal Transduction; Thiazoles; Tumor Suppressor Protein p53

Targeting the Mdm2 oncoprotein by drugs has the potential of re-establishing p53 function and tumor suppression. However, Mdm2-antagonizing drug candidates, e. g. Nutlin-3a, often fail to abolish cancer cell growth sustainably. To overcome these limitations, we inhibited Mdm2 and simultaneously a second negative regulator of p53, the phosphatase Wip1/PPM1D. When combining Nutlin-3a with the Wip1 inhibitor GSK2830371 in the treatment of p53-proficient but not p53-deficient cells, we observed enhanced phosphorylation (Ser 15) and acetylation (Lys 382) of p53, increased expression of p53 target gene products, and synergistic inhibition of cell proliferation. Surprisingly, when testing the two compounds individually, largely distinct sets of genes were induced, as revealed by deep sequencing analysis of RNA. In contrast, the combination of both drugs led to an expression signature that largely comprised that of Nutlin-3a alone. Moreover, the combination of drugs, or the combination of Nutlin-3a with Wip1-depletion by siRNA, activated p53-responsive genes to a greater extent than either of the compounds alone. Simultaneous inhibition of Mdm2 and Wip1 enhanced cell senescence and G2/M accumulation. Taken together, the inhibition of Wip1 might fortify p53-mediated tumor suppression by Mdm2 antagonists.

Topics: Acetylation; Aminopyridines; Antineoplastic Combined Chemotherapy Protocols; Cell Proliferation; Cellular Senescence; Dipeptides; Dose-Response Relationship, Drug; Drug Synergism; Enzyme Inhibitors; G2 Phase Cell Cycle Checkpoints; Gene Expression Regulation, Neoplastic; HCT116 Cells; Humans; Imidazoles; MCF-7 Cells; Neoplasms; Phosphorylation; Piperazines; Protein Phosphatase 2C; Proto-Oncogene Proteins c-mdm2; RNA Interference; Signal Transduction; Time Factors; Transcriptome; Transfection; Tumor Suppressor Protein p53; Up-Regulation

Regioselective synthesis, biological evaluation and 3D-molecular modeling for a series of novel diastereomeric 2-thioxo-5H-dispiro[imidazolidine-4,3-pyrrolidine-2,3-indole]-2,5(1H)-diones are described. The studied compounds have been tentatively identified as potent small molecule MDM2/p53 PPI inhibitors and can therefore be reasonably regarded as promising anticancer therapeutics.

Topics: Antineoplastic Agents; Cell Proliferation; Drug Design; Humans; Models, Molecular; Molecular Dynamics Simulation; Molecular Structure; Neoplasms; Proto-Oncogene Proteins c-mdm2; Small Molecule Libraries; Structure-Activity Relationship; Tumor Cells, Cultured; Tumor Suppressor Protein p53

The p53 tumor suppressor is the central component of a complex network of signaling pathways that protect organisms against the propagation of cells carrying oncogenic mutations. Here we report a previously unrecognized role of p53 in membrane phospholipids composition. By repressing the expression of stearoyl-CoA desaturase 1, SCD, the enzyme that converts saturated to mono-unsaturated fatty acids, p53 causes a shift in the content of phospholipids with mono-unsaturated acyl chains towards more saturated phospholipid species, particularly of the phosphatidylinositol headgroup class. This shift affects levels of phosphatidylinositol phosphates, attenuates the oncogenic AKT pathway, and contributes to the p53-mediated control of cell survival. These findings expand the p53 network to phospholipid metabolism and uncover a new molecular pathway connecting p53 to AKT signaling.

Topics: Animals; Cell Line, Tumor; Cell Survival; Cyclin-Dependent Kinase Inhibitor p21; Disease Progression; Fatty Acids, Unsaturated; Gene Expression Profiling; Gene Expression Regulation, Neoplastic; Humans; Imidazoles; Lipids; Male; Mice; Mice, Inbred C57BL; Mice, Transgenic; Neoplasms; Phospholipids; Piperazines; Proto-Oncogene Proteins c-akt; Proto-Oncogene Proteins c-mdm2; Signal Transduction; Stearoyl-CoA Desaturase; Sterol Regulatory Element Binding Protein 1; Tumor Suppressor Protein p53

We previously reported the discovery of a class of spirooxindoles as potent and selective small-molecule inhibitors of the MDM2-p53 interaction (MDM2 inhibitors). We report herein our efforts to improve their pharmacokinetic properties and in vivo antitumor activity. Our efforts led to the identification of 9 (MI-888) as a potent MDM2 inhibitor (Ki = 0.44 nM) with a superior pharmacokinetic profile and enhanced in vivo efficacy. Compound 9 is capable of achieving rapid, complete, and durable tumor regression in two types of xenograft models of human cancer with oral administration and represents the most potent and efficacious MDM2 inhibitor reported to date.

Topics: Animals; Area Under Curve; Blotting, Western; Cell Line, Tumor; Cell Proliferation; HCT116 Cells; Humans; Indoles; Inhibitory Concentration 50; Kinetics; Mice; Microsomes; Molecular Structure; Neoplasms; Oxindoles; Protein Binding; Proto-Oncogene Proteins c-mdm2; Rats; Rats, Sprague-Dawley; Spiro Compounds; Tumor Suppressor Protein p53; Xenograft Model Antitumor Assays

Derivatives with scaffolds of 1,3,5-tri-substituted pyrazoline and 1,3,4,5-tetra-substituted pyrazoline were synthesized and tested for their inhibitory effects versus the p53(+/+) HCT116 and p53(-/-) H1299 human tumor cell lines. Several compounds were active against the two cell lines displaying IC50 values in the low micromolar range with a clearly more pronounced effect on the p53(+/+) HCT116 cells. The compound class shows excellent developability due to the modular synthesis, allowing independent optimization of all three to four key substituents to improve the properties of the molecules.

Topics: Antineoplastic Agents; Cell Line, Tumor; Gene Deletion; Gene Expression Regulation, Neoplastic; Growth Inhibitors; Humans; Neoplasms; Structure-Activity Relationship; Tumor Suppressor Protein p53

Tumour cell-selective activation of apoptosis by recombinant human TNF-related apoptosis-inducing ligand (rhTRAIL) is enhanced through co-activation of p53 by chemotherapeutic drugs. The novel anticancer agent nutlin-3 provides a promising alternative for p53 activation by disrupting the interaction between p53 and its negative feedback regulator MDM2.. We examined whether nutlin-3 enhances apoptosis induction by rhTRAIL and the DR5-selective TRAIL variant D269H/E195R in wild-type p53-expressing ovarian, colon and lung cancer cell lines and in an ex vivo model of human ovarian cancer.. Nutlin-3 enhanced p53, p21, MDM2 and DR5 surface expression. Although nutlin-3 did not induce apoptosis, it preferentially enhanced D269H/E195R-induced apoptosis over rhTRAIL. Combination treatment potentiated the cleavage of caspases 8, 9, 3 and PARP. P53 and MDM2 siRNA experiments showed that this enhanced apoptotic effect was mediated by wild-type p53. Indeed, nutlin-3 did not enhance rhTRAIL-induced apoptosis in OVCAR-3 cells harbouring mutant p53. Addition of the chemotherapeutic drug cisplatin to the combination further increased p53 and DR5 levels and rhTRAIL- and D269H/E195R-induced apoptosis. As a proof of concept, we show that the combination of D269H/E195R, nutlin-3 and cisplatin induced massive apoptosis in ex vivo tissue slices of primary human ovarian cancers.. Nutlin-3 is a potent enhancer of D269H/E195R-induced apoptosis in wild-type p53-expressing cancer cells. Addition of DNA-damaging agents such as cisplatin further enhances DR5-mediated apoptosis.

Topics: Amino Acid Substitution; Apoptosis; Drug Resistance, Neoplasm; Drug Synergism; Genes, p53; Humans; Imidazoles; Neoplasms; Piperazines; Receptors, TNF-Related Apoptosis-Inducing Ligand; Recombinant Proteins; Substrate Specificity; TNF-Related Apoptosis-Inducing Ligand; Tumor Cells, Cultured

Cancer is characterized by uncontrolled growth of abnormal cells leading to the formation of tumors. Normally, the pro-apoptotic p53 protein plays a central role in protecting cells against carcinogenesis. In almost 50% human tumor cells, however, the p53 protein is dysregulated by direct interaction with its negative regulator, the Murine Double Minute 2 (MDM2) protein. Therefore, blocking the p53-binding pocket on MDM2, leading to the activation of tumor suppressor p53 presents a novel therapeutic strategy against several types of cancers. The published crystal structure of MDM2 bound with the p53 binding domain has revealed that three key hydrophobic residues of p53 are buried deep into the binding cavity of MDM2 and thus are central to p53-MDM2 binding. Accordingly, several low-molecular-weight compounds have been developed that mimic these three hydrophobic residues and thus bind to the MDM2 pocket, leading in turn to inhibition of the deleterious p53-MDM2 interaction. It is noteworthy that these inhibitors also possess an additional hydrophilic group that is shown to be necessary as a "cover" protecting the hydrophobic interaction surface between inhibitor and MDM2 from surrounding solvent. In comparison, several FDA-approved drugs possess the three key hydrophobic features necessary for binding to MDM2, but lack the fourth hydrophilic moiety, thus possibly hindering their ability as potential p53-MDM2 interaction inhibitors. Therefore, we hypothesize that conjugation of hydrophilic vitamin folic acid or its analogs to these drugs (termed "FOLICation") may provide them with the much-needed hydrophilic cover and make them suitable for investigation as potentially novel p53-MDM2 inhibitors. We also anticipate that FOLICation of these drugs may further lead to their enhanced and selective uptake by cancer cells, owing to the significantly higher expression of folic acid receptors on cancer cells compared to normal cells.

Topics: Clofazimine; Cyclopentanes; Drug Discovery; Folic Acid; Humans; Imidazoles; Indoles; Models, Molecular; Neoplasms; Piperazines; Protein Interaction Domains and Motifs; Proto-Oncogene Proteins c-mdm2; Spiro Compounds; Tumor Suppressor Protein p53

Pharmacological modulation of p53 activity is an attractive therapeutic strategy in cancers with wild-type p53. Presently in clinical trials, the small molecule Nutlin-3A competitively binds to HDM2, a key negative regulator of p53 and blocks its activity. We have described resistance mutations in HDM2 that selectively reduce affinity for Nutlin but not p53. In the present communication, we show that stapled peptides targeting the same region of HDM2 as Nutlin are refractory to these mutations, and display reduced discrimination between the wild-type and mutant HDM2s with regards to functional abrogation of interaction with p53. The larger interaction footprint afforded by stapled peptides suggests that this class of ligands may prove comparatively more resilient to acquired resistance in a clinical setting.

Topics: Animals; Cell Line; Drug Resistance, Neoplasm; Gene Knockout Techniques; Humans; Imidazoles; Ligands; Mice; Molecular Docking Simulation; Molecular Dynamics Simulation; Mutation; Neoplasms; Peptides; Piperazines; Protein Binding; Protein Interaction Domains and Motifs; Proto-Oncogene Proteins c-mdm2; Tumor Suppressor Protein p53

Nutlin3, a non-genotoxic agonist of p53, is currently in phase II clinical trials for cancer treatment. However, its effects on normal tissues and cell types remain largely to be determined. Drugs that can selectively target cancer cells as well as cooperate with the p53 pathway are thus greatly needed. Iron-superoxide dismutase (Fe-SOD) is a potential candidate as it selectively targets cancer cells by eliminating the abnormally high levels of reactive oxygen species (ROS) in cancer cells; it also inhibits cancer cell growth by induction of p27. Here, we show evidence that modulating redox and ROS homeostasis cooperates with Nutlin3 to selectively inhibit cancer cells in vitro and in vivo. Co-treatment of Fe-SOD and Nutlin3 showed synergistic inhibition on cancer cells in vitro, and the induction of p27 appeared to be involved. No effects were observed on normal cells. In addition, such co-treatment further exhibited synergistic inhibition on tumor growth in vivo in a murine B16 xenograft model, while the individual treatments only achieved very limited inhibition. Thus, Fe-SOD cooperated with Nutlin3 to selectively inhibit cancer cells in vitro and in vivo.

Topics: Animals; Cell Proliferation; Cyclin-Dependent Kinase Inhibitor p27; Drug Synergism; Female; Hep G2 Cells; Humans; Imidazoles; Liposomes; MCF-7 Cells; Melanoma, Experimental; Mice; Neoplasms; Piperazines; Reactive Oxygen Species; Skin Neoplasms; Superoxide Dismutase; Tumor Suppressor Protein p53; Xenograft Model Antitumor Assays

Starting with Nutlins as an initial lead, we designed and generated bicyclic scaffolds aiming to place cis-bischlorophenyl moiety at the equivalent location where the hydrophobic interaction with MDM2 could be expected. As a result, we discovered novel MDM2 inhibitors possessing a dihydroimidazothiazole scaffold. Further exploration of the side chains on the dihydroimidazothiazole scaffold aided by molecular modeling resulted in compounds exhibiting almost comparable in vitro potency to Nutlin-3a.

Topics: Drug Design; Humans; Imidazoles; Neoplasms; Protein Interaction Maps; Proto-Oncogene Proteins c-mdm2; Thiazoles; Tumor Suppressor Protein p53

The p53-MDM2 interaction has been proved to be a valuable target to develop effective antitumor agents. Novel p53-MDM2 inhibitors bearing pyrrolidone scaffolds were successfully identified by structure-based design. The nanomolar inhibitor 5 possessed good p53-MDM2 inhibitory activity (K(i) = 780 nM) due to its hydrophobic and hydrogen bonding interactions with MDM2. Further hit optimization led to the discovery of a number of highly potent pyrrolidone derivatives with improved p53-MDM2 inhibitory activity and in vitro antiproliferative potency. Compounds 41 (K(i) = 260.0 nM) and 60a (K(i) = 150.0 nM) showed good and selective activity against tumor cells with deleted p53. In addition, these two compounds also effectively inhibited the tumor growth in the A549 xenograft model. Interestingly, compound 41 was proved to be a potent MDM2/MDMX dual inhibitor. The novel pyrrolidone p53-MDM2 inhibitors represent promising lead structures for the development of novel antitumor agents.

Topics: Administration, Oral; Animals; Antineoplastic Agents; Apoptosis; Blotting, Western; Cell Cycle Proteins; Cell Proliferation; Humans; Imidazoles; Male; Mice; Mice, Inbred BALB C; Mice, Nude; Models, Molecular; Neoplasms; Nuclear Proteins; Protein Binding; Proto-Oncogene Proteins; Proto-Oncogene Proteins c-mdm2; Pyrrolidinones; Structure-Activity Relationship; Tumor Suppressor Protein p53

Nutlin-3, a small-molecule MDM2 inhibitor, restores p53 function and is, thus, an appealing candidate for the treatment of cancers retaining wild-type p53. However, nutlin-3 applied as single agent may be insufficient for cancer therapy. Therefore, we explored whether the anticancer activity of nutlin-3 could be enhanced by combination with histone deacetylase inhibitors (HDACi), i.e. vorinostat, sodium butyrate, MS-275 and apicidin. We found that nutlin-3 and HDACi cooperated to induce cell death in the p53 wild-type cell lines A549 and A2780, but not in the p53 null cell line PC-3, as assessed by Alamar Blue assay and flow cytometric analyses of propidium iodide uptake and mitochondrial depolarization. Combination index analysis showed that the effect was synergistic. For comparison, we tested nutlin-3 in combination with paclitaxel, revealing that nutlin-3 antagonized the cytotoxic activity of paclitaxel. To shed light on the underlying mechanism of the synergistic action of nutlin-3 and HDACi, we determined the acetylation status of p53 by immunoblotting and the mRNA levels of MDM2 and MDM4 by real-time RT-PCR. We observed vorinostat to induce p53 hyperacetylation, to reduce the constitutive gene expression of MDM2 and MDM4, and to counteract the nutlin-3-induced upregulation of MDM2 gene expression. In conclusion, our study shows that HDACi amplify the antitumor activity of nutlin-3-possibly by inducing p53 hyperacetylation and/or MDM2 and/or MDM4 downregulation-suggesting that treatment with a combination of nutlin-3 and HDACi may be an effective strategy for treating tumors with wild-type p53.

Topics: Acetylation; Antineoplastic Combined Chemotherapy Protocols; Benzamides; Blotting, Western; Butyrates; Cell Cycle Proteins; Cell Death; Cell Line, Tumor; Dose-Response Relationship, Drug; Down-Regulation; Drug Synergism; Flow Cytometry; Gene Expression Regulation, Enzymologic; Gene Expression Regulation, Neoplastic; Histone Deacetylase Inhibitors; Humans; Hydroxamic Acids; Imidazoles; Neoplasms; Nuclear Proteins; Paclitaxel; Peptides, Cyclic; Piperazines; Proto-Oncogene Proteins; Proto-Oncogene Proteins c-mdm2; Pyridines; Real-Time Polymerase Chain Reaction; RNA, Messenger; Tumor Suppressor Protein p53; Vorinostat

Mdm2 is the major negative regulator of p53 tumor-suppressor activity. This oncoprotein is overexpressed in many human tumors that retain the wild-type p53 allele. As such, targeted inhibition of Mdm2 is being considered as a therapeutic anticancer strategy. The N-terminal hydrophobic pocket of Mdm2 binds to p53 and thereby inhibits the transcription of p53 target genes. Additionally, the C-terminus of Mdm2 contains a RING domain with intrinsic ubiquitin E3 ligase activity. By recruiting E2 ubiquitin-conjugating enzyme(s), Mdm2 acts as a molecular scaffold to facilitate p53 ubiquitination and proteasome-dependent degradation. Mdmx (Mdm4), an Mdm2 homolog, also has a RING domain and hetero-oligomerizes with Mdm2 to stimulate its E3 ligase activity. Recent studies have shown that C-terminal residues adjacent to the RING domain of both Mdm2 and Mdmx contribute to Mdm2 E3 ligase activity. However, the molecular mechanisms mediating this process remain unclear, and the biological consequences of inhibiting Mdm2/Mdmx co-operation or blocking Mdm2 ligase function are relatively unexplored. This study presents biochemical and cell biological data that further elucidate the mechanisms by which Mdm2 and Mdmx co-operate to regulate p53 level and activity. We use chemical and genetic approaches to demonstrate that functional inhibition of Mdm2 ubiquitin ligase activity is insufficient for p53 activation. This unexpected result suggests that concomitant treatment with Mdm2/Mdmx antagonists may be needed to achieve therapeutic benefit.

Topics: Cell Cycle Proteins; Cell Line, Tumor; Cell Survival; Gene Expression; Humans; Imidazoles; Mutation; Neoplasms; Nuclear Proteins; Piperazines; Protein Binding; Protein Processing, Post-Translational; Protein Stability; Proto-Oncogene Proteins; Proto-Oncogene Proteins c-mdm2; Transcriptional Activation; Tumor Suppressor Protein p53; Ubiquitination

Several proteins have been suggested in promoting tumor formation in numerous human tissues by inactivating the tumor suppressor p53. This has generated interest in the development of small molecules to block these inhibitors of p53 and to regain p53 activity. Recently, we identified a small molecule, Inauhzin, which can inhibit SIRT1 activity and activate p53. SIRT1 is a deacetylase that deacetylates p53 and facilitates Mdm2 mediated p53 destabilization. In this study, we tested if combining Inauhzin with Nutlin-3, an inhibitor of MDM2-p53 binding, might synergistically activate p53 to suppress tumor growth. Indeed, at lower doses, combination of Inauhzin and Nutlin-3 exhibited a synergistic effect on inhibiting cell growth and promoting apoptosis in human colon and lung cancer cell lines in a p53-dependent fashion. Minimal effects were observed with treatment of either compound alone. Using a xenograft tumor model, we also showed a synergistic effect with both compounds. Thus, to fully regain p53 activity, targeting its multiple inhibitory proteins might be a better approach. Our study provides evidence supporting this concept for achieving better therapeutic efficacy in tumors that possess wild type p53.

Topics: Animals; Apoptosis; Cell Line, Tumor; Cell Proliferation; Drug Synergism; Female; Humans; Imidazoles; Indoles; Mice; Mice, SCID; Models, Biological; Neoplasms; Phenothiazines; Piperazines; Tumor Burden; Tumor Suppressor Protein p53; Xenograft Model Antitumor Assays

Recently much attention has been given to the anti-cancer drug nutlin-3a, an antagonist of murine double minute 2 (MDM2) that actively inhibits p53-MDM2 interaction. Reactivating p53 function by nutlin-3a thus provides a promising therapeutic strategy for the treatment of cancer. Although nutlin-3a seems a potential candidate in restoring p53 activity, it has many lacunae, toxicity, poor bioavailability, nonspecific delivery, and most importantly it is a substrate of multidrug resistance protein. The objective of the present study is to prepare and characterize nutlin-3a loaded poly(lactide-co-glycolide) (PLGA) nanoparticles (NPs), surface functionalized with epithelial cell adhesion molecule (EpCAM) antibody, with an aim to deliver encapsulated drug in a targeted manner to its site of action and to enhance its therapeutic efficacy many times over. The enhanced cellular uptake of EpCAM antibody conjugated nutlin-3a loaded NPs (EpCAM-nutlin-3a-NPs) over native nulin-3a, nutlin-3a loaded NPs (nutlin-3a-NPs) in HCT116 and A549 cells substantiate the targeting potentiality of conjugated system. IC₅₀ values depicted superior antiproliferative activity of EpCAM-nutlin-3a-NPs over nutlin-3a-NPs and native nutlin-3a in the above studied cell lines. Cell cycle arrest, loss of mitochondrial membrane potential and apoptosis induced by above formulation were confirmed by flow cytometry. Expression of p53, p21, EpCAM, and C-myc proteins involved in cell cycle regulation and apoptosis were investigated by western blotting. The above investigation indicates the enhanced therapeutic ability of EpCAM-nutlin-3a-NPs compared to nutlin-3a or nutlin-3a-NPs. Thus, our results suggest that EpCAM-nutlin-3a-NPs could be a potentially useful drug carrier system for targeted delivery of potent anti-cancer drug nutlin-3a for cancer therapy.

Topics: Antigens, Neoplasm; Blotting, Western; Cell Adhesion Molecules; Cell Cycle; Cell Death; Cell Line, Tumor; Chemical Phenomena; Coumarins; Epithelial Cell Adhesion Molecule; Humans; Imidazoles; Inhibitory Concentration 50; Intracellular Space; Membrane Potential, Mitochondrial; Microscopy, Confocal; Nanoparticles; Neoplasms; Particle Size; Piperazines; Polyglactin 910; Receptors, Cell Surface; Solutions

Topics: Antineoplastic Agents; Breast Neoplasms; Catalysis; Female; Humans; Imidazoles; Isomerism; Neoplasms; Ossification, Heterotopic; Piperazines; Proto-Oncogene Proteins c-mdm2; Receptors, Retinoic Acid; Retinoic Acid Receptor gamma; Tumor Suppressor Protein p53; Unfolded Protein Response

Chemotherapeutics (e.g., aurora kinase inhibitors) designed to target proliferative cells are often nonspecific for tumor cells as normal cycling cells are also susceptible. Indeed, one of the major dose-limiting toxicities of aurora kinase inhibitors is a dangerous depletion of neutrophils in patients. In this study we proposed a strategy to selectively target p53 mutant cells while sparing normal ones. The strategy is based on the understanding that normal cells have an intact p53 pathway but not tumor cells carrying p53 mutations. Nongenotoxic activation of p53 using nutlin led to a reversible activation of G1 and G2 arrest in normal cells, which prevents them from entering mitosis, thus protecting them from the side effects of aurora kinase inhibition (VX-680), namely endoreduplication and apoptosis. Cells carrying mutant p53 are selectively killed by the nutlin/VX-680 combination, whereas p53 wild-type cells retain their proliferative capacity. The major implications drawn from these results are: (1) reversible nongenotoxic activation of p53 may be used as a strategy for the chemoprotection of normal tissues, and (2) aurora kinase inhibitors may have alleviated side effects when used in combination with nutlin-like inhibitors. We highlight the distinct roles of p53 and p73 in mediating the cellular responses to VX-680 and suggest that dual protection by p53 and p73 are needed to guard against endoreduplication and polyploidy.

Topics: Apoptosis; Aurora Kinases; Caffeine; Cell Cycle; Cell Cycle Proteins; Cell Line; Cell Line, Tumor; Cell Proliferation; Coculture Techniques; Cyclin A2; Cyclin B1; Cyclin-Dependent Kinase Inhibitor p21; DNA-Binding Proteins; G2 Phase; Gene Expression; Humans; Imidazoles; Mutation; Neoplasms; Nuclear Proteins; Piperazines; Polyploidy; Protein Kinase Inhibitors; Protein Serine-Threonine Kinases; Proto-Oncogene Proteins c-mdm2; RNA, Small Interfering; Tetraploidy; Tumor Protein p73; Tumor Suppressor Protein p53; Tumor Suppressor Proteins

When the cell cycle is arrested, growth-promoting pathways such as mTOR (Target of Rapamycin) drive cellular senescence, characterized by cellular hyper-activation, hypertrophy and permanent loss of the proliferative potential. While arresting cell cycle, p53 (under certain conditions) can inhibit the mTOR pathway. Senescence occurs when p53 fails to inhibit mTOR. Low concentrations of DNA-damaging drugs induce p53 at levels that do not inhibit mTOR, thus causing senescence. In quiescence caused by serum starvation, mTOR is deactivated. This predicts that induction of p53 will not cause senescence in such quiescent cells. Here we tested this prediction. In proliferating normal cells, etoposide caused senescence (cells could not resume proliferation after removal of etoposide). Serum starvation prevented induction of senescence, but not of p53, by etoposide. When etoposide was removed, such cells resumed proliferation upon addition of serum. Also, doxorubicin did not cause senescent morphology in the absence of serum. Re-addition of serum caused mTOR-dependent senescence in the presence of etoposide or doxorubicin. Also, serum-starvation prevented senescent morphology caused by nutlin-3a in MCF-7 and Mel-10 cells. We conclude that induction of p53 does not activate the senescence program in quiescent cells. In cells with induced p53, re-activation of mTOR by serum stimulation causes senescence, as an equivalent of cellular growth.

Topics: Cell Cycle; Cell Line; Cell Proliferation; Cell Shape; Cellular Senescence; DNA Damage; Doxorubicin; Enzyme Activation; Etoposide; Humans; Imidazoles; Neoplasms; Piperazines; Protein Kinase Inhibitors; Retinal Pigment Epithelium; Serum; Sirolimus; Time Factors; TOR Serine-Threonine Kinases; Tumor Suppressor Protein p53; Up-Regulation

p53-Based cyclotherapy is proving to be a promising approach to palliate undesired effects of chemotherapy in patients with tumours carrying p53 mutations. For example, pre-treatment of cell cultures with Nutlin-3, a highly-selective inhibitor of the p53-mdm2 interaction, has been successfully used as a cytostatic agent to protect normal cells, but not p53-defective cells, from subsequent treatment with mitotic poisons or S-phase specific drugs. Here we sought to evaluate whether low doses of Actinomycin D (LDActD), a clinically-approved drug and potent p53 activator, could substitute Nutlin-3 in p53-based cyclotherapy. We found that pre-treatment with LDActD before adding the aurora kinase inhibitor VX-680 protects normal fibroblasts from polyploidy and nuclear morphology abnormalities induced by VX-680. However, and although to a lower extent than normal fibroblasts, tumour cell lines bearing p53 mutations were also protected by LDActD (but not Nutlin-3) from VX-680-induced polyploidy. We also report that a difference between the response of p53 wild-type cells and p53-defective cells to the LDActD/VX-680 sequential combination is that only the former fail to enter S-phase and therefore accumulate in G1/G0. We propose that drugs that incorporate into DNA during S-phase may perform better as second drugs than mitotic poisons in cyclotherapy approaches using LDActD as a cytostatic agent.

Topics: Aneugens; Aneuploidy; Antineoplastic Combined Chemotherapy Protocols; Aurora Kinases; Cell Cycle; Cell Line, Tumor; Cell Proliferation; Cytoprotection; Dactinomycin; Dose-Response Relationship, Drug; Gene Knockout Techniques; Genes, p53; HCT116 Cells; Humans; Imidazoles; Neoplasms; Piperazines; Protein Serine-Threonine Kinases

Through targeted homologous recombination, we developed a panel of matched colorectal cancer cell lines that differ only with respect to their endogenous TP53 status. We then used these lines to define the genes whose expression was altered after DNA damage induced by ionizing radiation. Transcriptome analyses revealed a consistent up-regulation of polo-like kinase 1 (PLK1) as well as other genes controlling the G(2)/M transition in the cells whose TP53 genes were inactivated compared with those with WT TP53 genes. This led to the hypothesis that the viability of stressed cells without WT TP53 depended on PLK1. This hypothesis was validated by demonstrating that stressed cancer cells without WT TP53 alleles were highly sensitive to PLK1 inhibitors, both in vivo and in vitro.

Topics: Alleles; Animals; Antineoplastic Agents; Cell Line, Tumor; Cyclin-Dependent Kinase Inhibitor p21; G1 Phase; Gene Expression Profiling; Gene Targeting; Genotype; Humans; Imidazoles; Mice; Neoplasms; Piperazines; Pteridines; Transcription, Genetic; Tumor Suppressor Protein p53; Xenograft Model Antitumor Assays

Cellular senescence is a stress-induced state of irreversible growth arrest thought to act as a barrier to cancer development. The p53 tumor suppressor is a critical mediator of senescence and recent in vivo studies have suggested that p53-induced senescence may contribute to tumor clearance by the immune system. Recently developed MDM2 antagonists, the nutlins, are effective p53 activators and potent antitumor agents in cells with functional apoptotic pathways. However, they only block cell cycle progression in cancer cells with compromised p53 apoptotic signaling. We use nutlin-3a as a selective probe to study the role of p53 activation in senescence using a panel of eight epithelial cancer cell lines and primary epithelial cells. Our results reveal that the MDM2 antagonist can induce a senescence-like state in all tested cell lines, but it is reversible and cells resume proliferation upon drug removal and normalization of p53 control. Retinoblastoma family members (pRb, p107, and p130) previously implicated in gene silencing during fibroblasts senescence were found down-regulated in cells with nutlin-induced senescence-like phenotype, suggesting a mechanism for its reversibility. Therefore, selective p53 pathway activation is insufficient for induction of true senescence in epithelial cells in vitro. However, elevated expression of several inflammatory cytokines in cancer cells with nutlin-induced senescence-like phenotype suggests a possible in vivo benefit of p53-activating therapies.

Topics: Apoptosis; Cell Cycle; Cell Growth Processes; Cell Line, Tumor; Cellular Senescence; Cytokines; Down-Regulation; Epithelial Cells; HCT116 Cells; Humans; Imidazoles; Neoplasms; Phenotype; Piperazines; Retinoblastoma Protein; Signal Transduction; Tumor Suppressor Protein p53

Nutlin-3 is a selective inhibitor of the p53-Mdm2 interaction, and inhibits growth in most tumor cells with wild-type p53. However, it only induces apoptosis in subsets of tumor cells. We report that the apoptotic response induced by Nutlin-3 correlates with its antitumor effects in xenograft models in athymic mice. We have investigated signals that sensitize cells to undergo apoptosis induced by Nutlin-3. We demonstrate that adenovirus E1A increases Nutlin-3-induced apoptosis through pRb inhibition in mouse embryonic fibroblast cells in a p53-dependent manner. Consistent with this, pRb depletion by siRNA transfection with Nutlin-3 synergistically increases apoptosis in HCT116 human colon cancer cells, which are insensitive to induction of apoptosis by Nutlin-3 alone. As pRb is a key negative regulator of E2F, we asked whether E2F transcriptional activity determines the apoptotic response of cancer cells to Nutlin-3. We demonstrate that transcriptional activity of E2F correlates with the apoptotic response to Nutlin-3 in various tumor cells and depletion of E2F-1 suppresses Nutlin-3-induced apoptosis in cells possessing high transcriptional activity of E2F, including retinoblastoma cells harboring mutated Rb with wild-type p53. Furthermore, we report that expression of the p53 homologue p73, a target of E2F-1, is markedly increased by Nutlin-3 in Rb-mutated tumor cells harboring wild-type p53. Depletion of p73 by siRNA transfection suppresses Nutlin-3-induced apoptosis in these cells. Taken together, our results demonstrate that E2F-1 transcriptional activity is a critical determinant of Mdm2 antagonist-induced apoptosis and p73 is important for E2F-1-mediated apoptosis induced by Nutlin-3, especially in tumor cells with mutated Rb. Furthermore, our results suggest that tumor cells, including Rb mutated cells, which harbor wild-type p53 and high E2F transcriptional activity, could be a good target for Mdm2 antagonist therapy.

Topics: Animals; Apoptosis; Cell Cycle Proteins; Cell Proliferation; Cells, Cultured; E2F1 Transcription Factor; G1 Phase; Humans; Imidazoles; Male; Mice; Mice, Inbred BALB C; Mice, Inbred NOD; Mice, Nude; Mutation; Neoplasms; Nuclear Proteins; Piperazines; Promoter Regions, Genetic; Proto-Oncogene Proteins; Proto-Oncogene Proteins c-mdm2; Resting Phase, Cell Cycle; Retinoblastoma Protein; RNA, Small Interfering; Stereoisomerism; Transcription, Genetic; Transfection; Tumor Cells, Cultured; Tumor Suppressor Protein p53

Mdm2 inhibits p53 transactivation by forming a p53-Mdm2 complex on chromatin. Upon DNA damage-induced complex disruption, such latent p53 can be activated, but in cells overexpressing Mdm2 because of a homozygous single nucleotide polymorphism at position 309 (T --> G) of mdm2, the complex is highly stable and cannot be disrupted by DNA damage, rendering p53 inactive.. To determine whether the p53 response phenotype is influenced differentially in cells with variable mdm2 genotypes, we compared responses to DNA damage and targeted p53-Mdm2 complex disruption by Nutlin-3 in the following wild-type p53 human cancer cell lines: A875 and CCF-STTG-1 (G/G for mdm2 SNP309), SJSA-1 (mdm2 genomic amplification and T/T for mdm2 SNP309), MCF-7 (estrogen-induced Mdm2 overexpression and T/G for mdm2 SNP309), ML-1 and H460 (T/T for mdm2 SNP309), and K562 (p53-null and T/G for mdm2 SNP309). We also examined mdm2 gene-splicing patterns in these lines by cloning and sequencing analyses.. While Mdm2-overexpressing G/G cells were resistant to p53 activation by DNA damage, they were sensitive to Nutlin-3. Strikingly, the p53 G1 checkpoint in G/G cells was activated by Nutlin-3 but not by etoposide, whereas in other Mdm2-overexpressing cells, both drugs activated p53 and subsequent G1 arrest or apoptosis. cDNA clones lacking exons 5-9 were generated at a high frequency in cells overexpressing Mdm2.. Nutlin-3 and DNA damage distinguish a differential phenotype in human cancer cells with G/G mdm2 SNP309 from other Mdm2 overexpressers. Categorization of the Mdm2 isoforms produced and their influence on p53 activity will help in characterization and treatment development for different cancers.

Topics: Antineoplastic Agents, Phytogenic; Cell Line, Tumor; Cloning, Molecular; DNA Damage; Etoposide; Flow Cytometry; Genotype; Humans; Imidazoles; Neoplasms; Phenotype; Piperazines; Polymorphism, Single Nucleotide; Proto-Oncogene Proteins c-mdm2; Reverse Transcriptase Polymerase Chain Reaction; Tumor Suppressor Protein p53

p53 Activity is controlled in large part by MDM2, an E3 ubiquitin ligase that binds p53 and promotes its degradation. The MDM2 antagonist Nutlin-3a stabilizes p53 by blocking its interaction with MDM2. Several studies have supported the potential use of Nutlin-3a in cancer therapy. Two different p53 wild-type cancer cell lines (U2OS and HCT116) treated with Nutlin-3a for 24 hours accumulated 2N and 4N DNA content, suggestive of G(1) and G(2) phase cell cycle arrest. This coincided with increased p53 and p21 expression, hypophosphorylation of pRb, and depletion of Cyclin B1, Cyclin A, and CDC2. Upon removal of Nutlin-3a, 4N cells entered S phase and re-replicated their DNA without an intervening mitotic division, a process known as endoreduplication. p53-p21 pathway activation was required for the depletion of Cyclin B1, Cyclin A, and CDC2 in Nutlin-3a-treated cells and for endoreduplication after Nutlin-3a removal. Stable tetraploid clones could be isolated from Nutlin-3a treated cells, and these tetraploid clones were more resistant to ionizing radiation and cisplatin-induced apoptosis than diploid counterparts. These data indicate that transient Nutlin-3a treatment of p53 wild-type cancer cells can promote endoreduplication and the generation of therapy-resistant tetraploid cells. These findings have important implications regarding the use of Nutlin-3a in cancer therapy

Topics: Apoptosis; CDC2 Protein Kinase; Cell Line, Tumor; Cyclin A; Cyclin B; Cyclin B1; Cyclin-Dependent Kinase Inhibitor p21; Cyclin-Dependent Kinases; DNA; DNA Damage; DNA Replication; Drug Resistance, Neoplasm; G1 Phase; G2 Phase; HCT116 Cells; Humans; Imidazoles; Neoplasms; Piperazines; Polyploidy; Tumor Suppressor Protein p53

microRNAs provide a novel layer of regulation for gene expression by interfering with the stability and/or translation of specific target mRNAs. Overall levels of microRNAs are frequently down-regulated in cancer cells, and reducing general microRNA processing increases cancerogenesis in transgenic models, suggesting that at least some microRNAs might act as effectors in tumor suppression. Accordingly, the tumor suppressor p53 up-regulates miR-34a, a microRNA that contributes to apoptosis and acute senescence. Here, we used array hybridization to find that p53 induces two additional, mutually related clusters of microRNAs, leading to the up-regulation of miR-192, miR-194, and miR-215. The same microRNAs were detected at high levels in normal colon tissue but were severely reduced in many colon cancer samples. On the other hand, miR-192 and its cousin miR-215 can each contribute to enhanced CDKN1A/p21 levels, colony suppression, cell cycle arrest, and cell detachment from a solid support. These effects were partially dependent on the presence of wild-type p53. Antagonizing endogenous miR-192 attenuated 5-fluorouracil-induced accumulation of p21. Hence, miR-192 and miR-215 can act as effectors as well as regulators of p53; they seem to suppress cancerogenesis through p21 accumulation and cell cycle arrest.

Topics: Bone Neoplasms; Cell Adhesion; Cell Cycle; Cell Line, Tumor; Colonic Neoplasms; Cyclin-Dependent Kinase Inhibitor p21; Genes, p53; HCT116 Cells; HT29 Cells; Humans; Imidazoles; MicroRNAs; Neoplasms; Oligonucleotide Array Sequence Analysis; Osteosarcoma; Piperazines; Proto-Oncogene Proteins c-mdm2; Transfection; Tumor Suppressor Protein p53; Up-Regulation

Nutlins, the newly developed small molecule antagonists of MDM2, activate p53 and induce apoptosis in cancer cells, offering a novel strategy of chemotherapy. Recent studies have further suggested synergistic effects of nutlins with other chemotherapeutic drugs. However, it is unclear whether nutlins increase or decrease the side effects of these drugs in normal non-malignant cells or tissues. Cisplatin is a widely used chemotherapy drug, which has a major side effect of kidney injury. Here we show that Nutlin-3 protected kidney cells against cisplatin-induced apoptosis. The cytoprotective effects of Nutlin-3 were not related to its regulation of p53 or consequent gene expression during cisplatin treatment. Moreover, the protective effects were shown in MDM2-, MDM4-, or p53-deficient cells. On the other hand, Nutlin-3 suppressed mitochondrial events of apoptosis during cisplatin incubation, including Bax activation and cytochrome c release. Nutlin-3 attenuated cisplatin-induced oligomerization of Bax and Bak but not their interactions with Bcl-XL. In isolated mitochondria, Nutlin-3 inhibited cytochrome c release induced by Ca2+, Bim peptide, and recombinant tBid. Importantly, it blocked both Bax and Bak oligomerization under these conditions. Together, the results have uncovered a new pharmacological function of nutlins, i.e. suppression of Bax and Bak, two critical mediators of apoptosis.

Topics: Animals; Antineoplastic Agents; Apoptosis; Apoptosis Regulatory Proteins; bcl-2 Homologous Antagonist-Killer Protein; bcl-2-Associated X Protein; Bcl-2-Like Protein 11; Calcium; Cell Line; Cisplatin; Cytochromes c; Drug Antagonism; Imidazoles; Kidney Tubules, Proximal; Membrane Proteins; Mitochondria; Neoplasms; Piperazines; Proto-Oncogene Proteins; Proto-Oncogene Proteins c-mdm2; Rats; Tumor Suppressor Protein p53

Strategies to treat cancer by restoring p53 tumor suppressor functions are being actively investigated. These approaches range from expressing an exogenous p53 gene in p53 mutant cancers to antagonizing a p53 inhibitor in p53 wild-type (WT) cancer cells. In addition, exogenous p53 is used to strengthen the anticancer efficacy of oncolytic adenoviruses. Many cancers express high levels of the major negative regulator of p53, mouse double minute 2 (MDM2) protein. Recently, a novel class of highly potent and specific MDM2 antagonists, the Nutlins, was identified. We envisioned that Nutlins could protect both endogenous and exogenous p53 from MDM2-mediated inactivation. We therefore investigated treating human cancer cells with a combination of adenovirus-mediated p53 gene therapy and Nutlin. Combination treatment resulted in broadly effective cell kill of p53 WT and p53-negative cancer cells. Cytotoxicity was associated with profound cell cycle checkpoint activation and apoptosis induction. We also tested Nutlin in combination with oncolytic adenoviruses. Nutlin treatment accelerated viral progeny burst from oncolytic adenovirus-infected cancer cells and caused an estimated 10- to 1,000-fold augmented eradication of p53 WT cancer cells. These findings suggest that Nutlins are promising compounds to be combined with p53 gene therapy and oncolytic virotherapy for cancer.

Topics: Adenoviridae; Apoptosis; Cell Count; Cell Line, Tumor; Flow Cytometry; Gene Expression Regulation, Neoplastic; Genes, p53; Genes, Reporter; Genotype; Humans; Imidazoles; Neoplasms; Oncolytic Viruses; Piperazines; Proto-Oncogene Proteins c-mdm2; Tumor Suppressor Protein p53

Current chemotherapy focuses on the use of genotoxic drugs that may induce general DNA damage in cancer cells but also high levels of toxicity in normal tissues. Nongenotoxic activation of p53 by targeting specific molecular pathways therefore provides an attractive therapeutic strategy in cancers with wild-type p53. Here, we explored the antitumor potential of cyclin-dependent kinase (CDK) inhibitors in combination with a small molecule inhibitor of p53-murine double minute 2 (MDM2) interaction. We show that low doses of CDK inhibitors roscovitine and DRB synergize with the MDM2 antagonist nutlin-3a in the induction of p53 activity and promote p53-dependent apoptosis in a dose- and time-dependent manner. Statistical measurement of the combination effects shows that the drug combination is additive on the reduction of cell viability and synergistic on inducing apoptosis, a critical end point of cytotoxic drugs. The degree of apoptosis observed 24 to 48 h after drug treatment correlated with the accumulation of p53 protein and concomitant induction of proapoptotic proteins Puma and PIG3. The antiproliferative and cytotoxic effects of this drug combination are validated in a range of tumor-derived cells including melanoma, colon carcinoma, breast adenocarcinoma, and hepatocarcinoma cells. Furthermore, this drug combination does not induce phosphorylation of Ser(15) on p53 and does not induce genotoxic stress in the cell. Given that many cytotoxic drugs rely on their ability to induce apoptosis via DNA damage-mediated activation of p53, the data presented here may provide a new therapeutic approach for the use of CDK inhibitors and MDM2 antagonists in combinatorial drug therapy.

Topics: Antineoplastic Agents; Antineoplastic Combined Chemotherapy Protocols; Apoptosis; Apoptosis Regulatory Proteins; Cell Line, Tumor; Cyclin-Dependent Kinases; Drug Synergism; Humans; Imidazoles; Intracellular Signaling Peptides and Proteins; Neoplasms; Piperazines; Proto-Oncogene Proteins; Proto-Oncogene Proteins c-mdm2; Purines; Roscovitine; Tumor Suppressor Protein p53

The identification of the cellular targets of small molecules with anticancer activity is crucial to their further development as drug candidates. Here, we present the application of a large-scale RNA interference-based short hairpin RNA (shRNA) barcode screen to gain insight in the mechanism of action of nutlin-3 (1). Nutlin-3 is a small-molecule inhibitor of MDM2, which can activate the p53 pathway. Nutlin-3 shows strong antitumor effects in mice, with surprisingly few side effects on normal tissues. Aside from p53, we here identify 53BP1 as a critical mediator of nutlin-3-induced cytotoxicity. 53BP1 is part of a signaling network induced by DNA damage that is frequently activated in cancer but not in healthy tissues. Our results suggest that nutlin-3's tumor specificity may result from its ability to turn a cancer cell-specific property (activated DNA damage signaling) into a weakness that can be exploited therapeutically.

Topics: Animals; Antineoplastic Agents; Blotting, Western; Cell Line, Tumor; Cells, Cultured; DNA Damage; Electronic Data Processing; Fibroblasts; Gene Expression Regulation, Neoplastic; Genes, p53; Genetic Techniques; Humans; Imidazoles; Intracellular Signaling Peptides and Proteins; Mice; Microscopy, Fluorescence; Models, Chemical; Neoplasms; Nuclear Proteins; Nucleic Acid Hybridization; Oligonucleotide Array Sequence Analysis; Phosphoproteins; Piperazines; Plasmids; Proto-Oncogene Proteins c-mdm2; RNA Interference; RNA, Small Interfering; Signal Transduction; Tumor Suppressor p53-Binding Protein 1; Tumor Suppressor Protein p53

The p53 tumor suppressor retains its wild-type conformation and transcriptional activity in half of all human tumors, and its activation may offer a therapeutic benefit. However, p53 function could be compromised by defective signaling in the p53 pathway. Using a small-molecule MDM2 antagonist, nutlin-3, to probe downstream p53 signaling we find that the cell-cycle arrest function of the p53 pathway is preserved in multiple tumor-derived cell lines expressing wild-type p53, but many have a reduced ability to undergo p53-dependent apoptosis. Gene array analysis revealed attenuated expression of multiple apoptosis-related genes. Cancer cells with mdm2 gene amplification were most sensitive to nutlin-3 in vitro and in vivo, suggesting that MDM2 overexpression may be the only abnormality in the p53 pathway of these cells. Nutlin-3 also showed good efficacy against tumors with normal MDM2 expression, suggesting that many of the patients with wild-type p53 tumors may benefit from antagonists of the p53-MDM2 interaction.

Topics: Animals; Antineoplastic Agents; Cell Cycle; Cell Line; Gene Expression Profiling; Gene Expression Regulation, Neoplastic; Humans; Imidazoles; Mice; Mice, Nude; Neoplasms; Piperazines; Proto-Oncogene Proteins c-mdm2; Signal Transduction; Tumor Suppressor Protein p53; Xenograft Model Antitumor Assays

Tumors that express wild-type P53 provide a target for therapies designed to reactivate P53 function. This is supported by the potent activation of P53 in tumor cells by Nutlin, a cis-imidazoline that inhibits the Hdm2-P53 interaction. The efficacy of Hdm2.P53 antagonists could be compromised if they do not antagonize Hdmx, an Hdm2 homolog that inhibits P53 transactivation. We evaluated the role of Hdmx expression in sensitivity to Nutlin in a range of cancer cell lines. Nutlin reduced Hdmx levels in normal cells and some cancer cell lines, whereas other cancer cells were refractory to such down-regulation. Strikingly, Nutlin did not disrupt Hdmx.P53 complexes, and in cell lines where no Hdmx degradation occurred, Nutlin failed to induce apoptosis. shRNA-mediated reduction of Hdmx sensitized cells to apoptosis, but caspase-3 was neither required nor sufficient for Hdmx degradation or apoptosis. Our data imply that Hdmx is an important determinant of the outcome of P53 activation. Thus, targeting Hdmx may be a therapeutic strategy that complements drugs such as Nutlin.

Topics: Apoptosis; Caspase 3; Cell Cycle Proteins; Cell Line, Tumor; Down-Regulation; Humans; Imidazoles; Neoplasms; Nuclear Proteins; Piperazines; Protein Binding; Proto-Oncogene Proteins; RNA Interference; Tumor Suppressor Protein p53