nutlin-3a and Breast-Neoplasms

Side effects of chemotherapy are a major impediment in the treatment of cancer. Cyclotherapy is an emerging therapeutic strategy for protecting normal cells from the side effects of chemotherapy. Low, non-genotoxic doses of known p53 activators can be used to induce p53-dependent cell cycle arrest in normal cells bearing wild-type p53. This cytostatic effect of p53 can protect normal cells from the toxicity of S- or M-phase poisons. Here, we have reviewed existing cyclotherapy regimens using two well-known p53 activators, nutlin-3 and actinomycin D. We have highlighted an exemplar clinical perspective for cyclotherapy in breast cancer. The recent development of novel stapled peptides as activators of p53 without the corresponding cytotoxicity holds great promise for cyclotherapy to enhance the therapeutic window of existing chemotherapy drugs.

Topics: Animals; Antineoplastic Combined Chemotherapy Protocols; Apoptosis; Breast Neoplasms; Cell Growth Processes; Cell Line, Tumor; Dactinomycin; Female; Genome; Humans; Imidazoles; Phosphorylation; Piperazines; Tumor Suppressor Protein p53

Despite an improvement of prognosis in breast and colon cancer, the outcome of the metastatic disease is still severe. Microevolution of cancer cells often leads to drug resistance and tumor-recurrence. To target the driving forces of the tumor microevolution, we focused on synergistic drug combinations of selected compounds. The aim is to prevent the tumor from evolving in order to stabilize disease remission. To identify synergisms in a high number of compounds, we propose here a three-step concept that is cost efficient, independent of high-throughput machines and reliable in its predictions.. We created dose response curves using MTT- and SRB-assays with 14 different compounds in MCF-7, HT-29 and MDA-MB-231 cells. In order to efficiently screen for synergies, we developed a screening tool in which 14 drugs were combined (91 combinations) in MCF-7 and HT-29 using EC. All 14 compounds exhibit antitumor effects on each of the three cell lines. The screening tool resulted in 19 potential synergisms detected in HT-29 (20.9%) and 27 in MCF-7 (29.7%). Seven of the top combinations were further verified over the whole dose response curve, and for five combinations a significant synergy could be confirmed. The combination Nutlin-3 (inhibition of MDM2) and PX-478 (inhibition of HIF-1α) could be confirmed for all three cell lines. The same accounts for the combination of Dichloroacetate (PDH activation) and NHI-2 (LDH-A inhibition). Our screening method proved to be an efficient tool that is reliable in its projections.. The presented three-step concept proved to be cost- and time-efficient with respect to the resulting data. The newly found combinations show promising results in MCF-7, HT-29 and MDA-MB231 cancer cells.

Topics: Antineoplastic Combined Chemotherapy Protocols; Breast Neoplasms; Cell Line, Tumor; Colonic Neoplasms; Dichloroacetic Acid; Drug Screening Assays, Antitumor; Drug Synergism; Female; Humans; Hypoxia-Inducible Factor 1, alpha Subunit; Imidazoles; L-Lactate Dehydrogenase; Mustard Compounds; Phenylpropionates; Piperazines; Proto-Oncogene Proteins c-mdm2; Pyruvate Dehydrogenase Complex; Reproducibility of Results

The identification of the essential role of cyclin-dependent kinases (CDKs) in the control of cell division has prompted the development of small-molecule CDK inhibitors as anticancer drugs. For many of these compounds, the precise mechanism of action in individual tumor types remains unclear as they simultaneously target different classes of CDKs - enzymes controlling the cell cycle progression as well as CDKs involved in the regulation of transcription. CDK inhibitors are also capable of activating p53 tumor suppressor in tumor cells retaining wild-type p53 gene by modulating MDM2 levels and activity. In the current study, we link, for the first time, CDK activity to the overexpression of the MDM4 (MDMX) oncogene in cancer cells. Small-molecule drugs targeting the CDK9 kinase, dinaciclib, flavopiridol, roscovitine, AT-7519, SNS-032, and DRB, diminished MDM4 levels and activated p53 in A375 melanoma and MCF7 breast carcinoma cells with only a limited effect on MDM2. These results suggest that MDM4, rather than MDM2, could be the primary transcriptional target of pharmacological CDK inhibitors in the p53 pathway. CDK9 inhibitor atuveciclib downregulated MDM4 and enhanced p53 activity induced by nutlin-3a, an inhibitor of p53-MDM2 interaction, and synergized with nutlin-3a in killing A375 melanoma cells. Furthermore, we found that human pluripotent stem cell lines express significant levels of MDM4, which are also maintained by CDK9 activity. In summary, we show that CDK9 activity is essential for the maintenance of high levels of MDM4 in human cells, and drugs targeting CDK9 might restore p53 tumor suppressor function in malignancies overexpressing MDM4.

Topics: Animals; Breast Neoplasms; Cell Cycle Proteins; Cell Line, Tumor; Cyclin-Dependent Kinase 9; Drug Synergism; Humans; Imidazoles; MCF-7 Cells; Melanoma; Mice; Piperazines; Pluripotent Stem Cells; Protein Kinase Inhibitors; Proto-Oncogene Proteins; Proto-Oncogene Proteins c-mdm2; Roscovitine; Sulfonamides; Transcription, Genetic; Transfection; Triazines

In a previous study, we reported that α‑smooth muscle actin (α‑SMA), one of the mesenchymal marker proteins, is highly expressed in tamoxifen‑resistant breast cancer (TamR) cells. However, the exact mechanism of α‑SMA expression in TamR cells is not fully understood. Here, we investigated the effect of TP53 on α‑SMA expression in breast cancer cells. The levels of α‑SMA mRNA and protein expression were analyzed by real‑time PCR and western blotting, respectively. In estrogen receptor‑positive [ER(+)] breast cancer patients, aberrant α‑SMA expression was found to be associated with a poor prognosis. The level of α‑SMA expression was significantly increased in established TamR cells compared to tamoxifen‑sensitive (TamS) cells. To verify the regulatory mechanism of α‑SMA expression, we analyzed diverse kinase activities between TamS and TamR cells. The activity of TP53 was markedly increased in the TamR cells. When TamS cells were treated with TP53 activator, Nutlin3 (Nut3), α‑SMA expression was increased in the TamS cells. In addition, α‑SMA expression was significantly increased by TP53 overexpression in breast cancer cells. On the contrary, the basal level of α‑SMA expression was decreased by the TP53 inhibitor, pifithrin‑α (PFT‑α). Taken together, we demonstrated that α‑SMA expression is regulated by TP53 activity in TamR cells.

Topics: Actins; Antineoplastic Agents, Hormonal; Breast Neoplasms; Datasets as Topic; Drug Resistance, Neoplasm; Female; Gene Expression Regulation, Neoplastic; Humans; Imidazoles; Kaplan-Meier Estimate; MCF-7 Cells; Piperazines; Prognosis; Receptors, Estrogen; Tamoxifen; Tumor Suppressor Protein p53; Up-Regulation

Alcohol consumption is associated with increased breast cancer risk; however, the underlying mechanisms that contribute to mammary tumor initiation and progression are unclear. Alcohol is known to induce oxidative stress and DNA damage; likewise, p53 is a critical modulator of the DNA repair pathway and ensures genomic integrity. p53 mutations are frequently detected in breast and other tumors. The impact of alcohol on p53 is recognized, yet the role of p53 in alcohol-induced mammary carcinogenesis remains poorly defined. In our study, we measured alcohol-mediated oxidative DNA damage in MCF-7 cells using 8-OHdG and p-H2AX foci formation assays. p53 activity and target gene expression after alcohol exposure were determined using p53 luciferase reporter assay, qPCR, and Western blotting. A mechanistic study delineating the role of p53 in DNA damage response and cell cycle arrest was based on isogenic MCF-7 cells stably transfected with control (MCF-7/Con) or p53-targeting siRNA (MCF-7/sip53), and MCF-7 cells that were pretreated with Nutlin-3 (Mdm2 inhibitor) to stabilize p53. Alcohol treatment resulted in significant DNA damage in MCF-7 cells, as indicated by increased levels of 8-OHdG and p-H2AX foci number. A p53-dependent signaling cascade was stimulated by alcohol-induced DNA damage. Moderate to high concentrations of alcohol (0.1-0.8% v/v) induced p53 activation, as indicated by increased p53 phosphorylation, reporter gene activity, and p21/Bax gene expression, which led to G0/G1 cell cycle arrest. Importantly, compared to MCF-7/Con cells, alcohol-induced DNA damage was significantly enhanced, while alcohol-induced p21/Bax expression and cell cycle arrest were attenuated in MCF-7/sip53 cells. In contrast, inhibition of p53 degradation via Nutlin-3 reinforced G0/G1 cell cycle arrest in MCF-7 control cells. Our study suggests that functional p53 plays a critical role in cellular responses to alcohol-induced DNA damage, which protects the cells from DNA damage associated with breast cancer risk.

Topics: 8-Hydroxy-2'-Deoxyguanosine; Alcohol Drinking; bcl-2-Associated X Protein; Breast Neoplasms; Cell Line, Tumor; Cell Transformation, Neoplastic; Cyclin-Dependent Kinase Inhibitor p21; Deoxyguanosine; DNA Damage; DNA Repair; Ethanol; G1 Phase Cell Cycle Checkpoints; Gene Expression Regulation, Neoplastic; Histones; Humans; Imidazoles; MCF-7 Cells; Oxidative Stress; Piperazines; Proto-Oncogene Proteins c-mdm2; RNA Interference; RNA, Small Interfering; Tumor Suppressor Protein p53

In breast cancer, p53 could be functionally compromised by interaction with several proteins. Among those proteins, MDM2 serves as a pivotal negative regulator and counteracts p53 activation. Thus, the ability to quantitatively and accurately monitor the changes in level of p53-MDM2 interaction with disease state can enable an improved understanding of this protein-protein interaction (PPI), provide a better insight into cancer development and allow the emergence of advanced treatments. However, rare studies have evaluated the quantitative extent of PPI including p53-MDM2 interaction so far. In this study, a LC-MS/MS-based targeted proteomics assay was developed and coupled with co-immunoprecipitation (Co-IP) for the quantification of p53-MDM2 complex. A p53 antibody with the epitope residing at 156-214 residues achieved the greatest IP efficiency. 321KPLDGEYFTLQIR333 (p53) and 327ENWLPEDK334 (MDM2) were selected as surrogate peptides in the targeted analysis. Stable isotope-labeled synthetic peptides were used as internal standards. An LOQ (limit of quantification) of 2ng/mL was obtained. Then, the assay was applied to quantitatively detect total p53, total MDM2 and p53-MDM2 in breast cells and tissue samples. Western blotting was performed for a comparison. Finally, a quantitative time-course analysis in MCF-7 cells with the treatment of nutlin-3 as a PPI inhibitor was also monitored.. Proteins do not function as single entities but rather as a team player that has to communicate. Protein-protein interaction (PPI), normally by means of non-covalent contact among binary or large protein complex, is essential for many cellular processes including cancer progression. Thus, the ability to quantitatively and accurately monitor the changes in level of PPI with disease state can enable an improved understanding of PPI, provide a better insight into cancer development and allow the emergence of advanced treatments. However, rare studies have evaluated the quantitative extent of PPI so far. The major issue of current available approaches is the trade-off between sensitivity and specificity. Thus, techniques with the ability to quantify PPIs with both high sensitivity (low false-negative rate) and high specificity (low false-positive rate) are eagerly desired. Liquid chromatography-tandem mass spectrometry (LC-MS/MS)-based targeted proteomics has shown its potential to study biomolecules because of its high sensitivity, high selectivity and wide dynamic range. In this study, we made an effort to develop a LC-MS/MS-based targeted proteomics assay for the quantitative detection of p53-MDM2 interaction in breast cells and tissue samples.

Topics: Biopsy; Breast Neoplasms; Cell Line, Tumor; Chromatography, Liquid; Humans; Imidazoles; MCF-7 Cells; Piperazines; Protein Interaction Maps; Proteomics; Proto-Oncogene Proteins c-mdm2; Tandem Mass Spectrometry; Tumor Suppressor Protein p53

The cell fate determinant Numb is frequently downregulated in human breast cancers (BCs), resulting in p53 inactivation and an aggressive disease course. In the mouse mammary gland, Numb/p53 downregulation leads to aberrant tissue morphogenesis, expansion of the stem cell compartment, and emergence of cancer stem cells (CSCs). Strikingly, CSC phenotypes in a Numb-knockout mouse model can be reverted by Numb/p53 restoration. Thus, targeting Numb/p53 dysfunction in Numb-deficient human BCs could represent a novel anti-CSC therapy. Here, using patient-derived xenografts, we show that expansion of the CSC pool, due to altered self-renewing divisions, is also a feature of Numb-deficient human BCs. In these cancers, using the inhibitor Nutlin-3 to restore p53, we corrected the defective self-renewal properties of Numb-deficient CSCs and inhibited CSC expansion, with a marked effect on tumorigenicity and metastasis. Remarkably, a regimen combining Nutlin-3 and chemotherapy induced persistent tumor growth inhibition, or even regression, and prevented CSC-driven tumor relapse after removal of chemotherapy. Our data provide a pre-clinical proof-of-concept that targeting Numb/p53 results in a specific anti-CSC therapy in human BCs.

Topics: Animals; Antineoplastic Agents, Phytogenic; Breast; Breast Neoplasms; Down-Regulation; Female; Humans; Imidazoles; Membrane Proteins; Mice; Mice, SCID; Neoplastic Stem Cells; Nerve Tissue Proteins; Paclitaxel; Piperazines; Tumor Cells, Cultured; Tumor Suppressor Protein p53

Breast cancer remains a significant health problem due to the involvement of multiple aberrant and redundant signaling pathways in tumorigenesis and the development of resistance to the existing therapeutic agents. Therefore, the search for novel chemotherapeutic agents for effective management of breast cancer is still warranted. In an effort to develop new anti-breast cancer agents, we have synthesized and identified novel spiro-oxindole derivative G613 i.e. 5-chloro-4',5'-diphenyl-3'-(4-(2-(piperidin-1-yl) ethoxy) benzoyl) spiro[indoline-3,2'-pyrrolidin]-2-one, which has shown growth inhibitory activity in breast cancer cells. The present study was aimed to explore the mechanism of anti-tumorigenic action of this newly identified spiro-oxindole compound. Compound G613 inhibited the Mdm2-p53 interaction in breast cancer cells and tumor xenograft. It caused restoration of p53 function by activating its promoter activity, triggering its nuclear accumulation and preventing its ubiquitination and proteasomal degradation. Supportively, molecular docking studies revealed considerable homology in the docking mode of G613 and the known Mdm2 inhibitor Nutlin-3, to p53 binding pocket of Mdm2. The activation of p53 led to upregulation of p53 dependent pro-apoptotic proteins, Bax, Pumaα and Noxa and enhanced interaction of p53 with bcl2 member proteins thus triggering both transcription-dependent and transcription-independent apoptosis, respectively. Additionally, the compound decreased estrogen receptor activity through sequestration of estrogen receptor α by p53 thereby causing a decreased transcriptional activation and expression of proliferation markers. In conclusion, G613 represents a potent small-molecule inhibitor of the Mdm2-p53 interaction and can serve as a promising lead for developing a new class of anti-cancer therapy for breast cancer patients.

Topics: Aged; Animals; Antineoplastic Agents; Apoptosis; Apoptosis Regulatory Proteins; bcl-2-Associated X Protein; Breast Neoplasms; Carcinoma, Ductal, Breast; Cell Proliferation; Chlorocebus aethiops; Female; Gene Expression Regulation, Neoplastic; HEK293 Cells; Humans; Imidazoles; Indoles; MCF-7 Cells; Mice; Mice, Nude; Middle Aged; Molecular Docking Simulation; Piperazines; Protein Binding; Proteolysis; Proto-Oncogene Proteins; Proto-Oncogene Proteins c-bcl-2; Proto-Oncogene Proteins c-mdm2; Spiro Compounds; Tumor Suppressor Protein p53; Vero Cells; Xenograft Model Antitumor Assays

Heat-shock protein 90 (Hsp90) is highly expressed in many tumor cells and is associated with the maintenance of malignant phenotypes. Targeting Hsp90 has had therapeutic success in both solid and hematological malignancies, which has inspired more studies to identify new Hsp90 inhibitors with improved clinical efficacy. Using a fragment-based approach and subsequent structural optimization guided by medicinal chemistry principles, we identified the novel compound CPUY201112 as a potent Hsp90 inhibitor. It binds to the ATP-binding pocket of Hsp90 with a kinetic dissociation (Kd) constant of 27 ± 2.3 nM. It also exhibits potent in vitro antiproliferative effects in a range of solid tumor cells. In MCF-7 cells with high Hsp90 expression, CPUY201112 induces the degradation of Hsp90 client proteins including HER-2, Akt, and c-RAF. We prove that treating MCF-7 cells with CPUY201112 results in cell cycle arrest and apoptosis through the wild-type (wt) p53 pathway. CPUY201112 also synergizes with Nutlin-3a to induce cancer cell apoptosis. CPUY201112 significantly inhibited the growth of MCF-7 xenografts in nude mice without apparent body weight loss. These results demonstrate that CPUY201112 is a novel Hsp90 inhibitor with potential use in treating wild-type p53 related cancers.

Topics: Adenocarcinoma; Animals; Antineoplastic Agents; Breast Neoplasms; Drug Synergism; Female; Gene Expression Regulation, Neoplastic; HSP90 Heat-Shock Proteins; Humans; Imidazoles; MCF-7 Cells; Mice; Mice, Nude; Piperazines; Proteolysis; Proto-Oncogene Proteins c-akt; Proto-Oncogene Proteins c-raf; Pyrimidines; Receptor, ErbB-2; Resorcinols; Signal Transduction; Tumor Burden; Tumor Suppressor Protein p53; Xenograft Model Antitumor Assays

PP2C family serine/threonine phosphatase WIP1 acts as a negative regulator of the tumor suppressor p53 and is implicated in silencing of cellular responses to genotoxic stress. Chromosomal locus 17q23 carrying the PPM1D (coding for WIP1) is commonly amplified in breast carcinomas and WIP1 was proposed as potential pharmacological target. Here we employed a cellular model with knocked out PPM1D to validate the specificity and efficiency of GSK2830371, novel small molecule inhibitor of WIP1. We have found that GSK2830371 increased activation of the DNA damage response pathway to a comparable level as the loss of PPM1D. In addition, GSK2830371 did not affect proliferation of cells lacking PPM1D but significantly supressed proliferation of breast cancer cells with amplified PPM1D. Over time cells treated with GSK2830371 accumulated in G1 and G2 phases of the cell cycle in a p21-dependent manner and were prone to induction of senescence by a low dose of MDM2 antagonist nutlin-3. In addition, combined treatment with GSK2830371 and doxorubicin or nutlin-3 potentiated cell death through a strong induction of p53 pathway and activation of caspase 9. We conclude that efficient inhibition of WIP1 by GSK2830371 sensitizes breast cancer cells with amplified PPM1D and wild type p53 to chemotherapy.

Topics: Aminopyridines; Apoptosis; Breast Neoplasms; Cell Cycle; Cell Proliferation; Dipeptides; DNA Damage; Drug Resistance, Neoplasm; Female; Humans; Imidazoles; Piperazines; Protein Phosphatase 2C; Proto-Oncogene Proteins c-mdm2; Tumor Cells, Cultured; Tumor Suppressor Protein p53

We report herein the design and synthesis of bioisosteres of spirooxindole (MI-63/219), a small-molecule inhibitors of the MDM2-p53 interaction as anti-breast cancer agents. Compound 5b has been exhibiting significant anti-proliferative activity in nude mice bearing MCF-7 xenograft tumor. The compound 5b was found to act via modulation of MDM2 and p53 expression in breast cancer cells expressing wild type p53. Compound 5b stimulated p53 activation, caused modulation of downstream effectors p21, pRb, and cyclin D1 which regulate cell cycle. Thus, compound triggered G1-S phase cell cycle arrest, which was evident by flow cytometric analysis of treated breast cancer cells. Thus, compound 5b restores the p53 function, which triggers molecular events consistent with cell cycle arrest at G1/S phase.

Topics: Animals; Antineoplastic Agents; Breast; Breast Neoplasms; Cell Line, Tumor; Drug Design; Female; Humans; Indoles; Mice, Nude; Oxindoles; Protein Interaction Maps; Proto-Oncogene Proteins c-mdm2; Spiro Compounds; Tumor Suppressor Protein p53

The promising proposition of multifunctional nanoparticles for cancer diagnostics and therapeutics has inspired the development of theranostic approach for improved cancer therapy. Moreover, active targeting of drug carrier to specific target site is crucial for providing efficient delivery of therapeutics and imaging agents. In this regard, the present study investigates the theranostic capabilities of nutlin-3a loaded poly (lactide-co-glycolide) nanoparticles, functionalized with a targeting ligand (EpCAM aptamer) and an imaging agent (quantum dots) for cancer therapy and bioimaging. A wide spectrum of in vitro analysis (cellular uptake study, cytotoxicity assay, cell cycle and apoptosis analysis, apoptosis associated proteins study) revealed superior therapeutic potentiality of targeted NPs over other formulations in EpCAM expressing cells. Moreover, our nanotheranostic system served as a superlative bio-imaging modality both in 2D monolayer culture and tumor spheroid model. Our result suggests that, these aptamer-guided multifunctional NPs may act as indispensable nanotheranostic approach toward cancer therapy.. This study investigated the theranostic capabilities of nutlin-3a loaded poly (lactide-co-glycolide) nanoparticles functionalized with a targeting ligand (EpCAM aptamer) and an imaging agent (quantum dots) for cancer therapy and bioimaging. It was concluded that the studied multifunctional targeted nanoparticle may become a viable and efficient approach in cancer therapy.

Topics: Antigens, Neoplasm; Apoptosis; Breast Neoplasms; Cell Adhesion Molecules; Drug Delivery Systems; Epithelial Cell Adhesion Molecule; Female; Humans; Imidazoles; Lactic Acid; Nanoparticles; Piperazines; Polyglycolic Acid; Polylactic Acid-Polyglycolic Acid Copolymer; Quantum Dots

Let-7 miRNAs act as tumour suppressors by directly binding to the 3'UTRs of downstream gene products. The regulatory role of let-7 in downstream gene expression has gained much interest in the cancer research community, as it controls multiple biological functions and determines cell fates. For example, one target of the let-7 family is cyclin D1, which promotes G0/S cell cycle progression and oncogenesis, was correlated with endoribonuclease DICER1, another target of let-7. Down-regulated let-7 has been identified in many types of tumours, suggesting a feedback loop may exist between let-7 and cyclin D1. A potential player in the proposed feedback relationship is Dicer, a central regulator of miRNA expression through sequence-specific silencing. We first identified that DICER1 is the key downstream gene for cyclin D1-induced let-7 expression. In addition, we found that let-7 miRNAs expression decreased because of the p53-induced cell death response, with deregulated cyclin D1. Our results also showed that cyclin D1 is required for Nutlin-3 and TAX-induced let-7 expression in cancer repression and the cell death response. For the first time, we provide evidence that let-7 and cyclin D1 form a feedback loop in regulating therapy response of cancer cells and cancer stem cells, and importantly, that alteration of let-7 expression, mainly caused by cyclin D1, is a sensitive indicator for better chemotherapies response.

Topics: 3' Untranslated Regions; Apoptosis; Breast Neoplasms; Cell Cycle; Cell Line, Tumor; Cell Survival; Cyclin D1; DEAD-box RNA Helicases; Female; Gene Expression Regulation, Neoplastic; Humans; Imidazoles; MCF-7 Cells; MicroRNAs; Microscopy, Fluorescence; Piperazines; Ribonuclease III; RNA Interference; Signal Transduction; Spheroids, Cellular; Tumor Suppressor Protein p53

Estrogen is synthesized from cholesterol and high cholesterol levels are suggested to be associated with increased risk of estrogen receptor(ER)-positive breast cancer. The cholesterol metabolite 27-hydroxycholesterol (27-OHC) was recently identified as a selective estrogen receptor modulator (SERM) and may therefore impact breast cancer progression. However, the mechanisms by which 27-OHC may contribute to breast cancer are not all known. We determined the extent to which 27-OHC regulates cell proliferation in MCF7 ER-positive breast cancer cell line involving the tumor suppressor protein p53. We found that treatment of MCF7 cells with 27-OHC resulted reduced p53 transcriptional activity. Conversely, treatment of the ER-negative MDA-MB 231 cells with 27-OHC induced no significant change in p53 activity. Exposure of MCF7 cells to 27-OHC was also associated with increased protein levels of the E3 ubiquitin protein ligase MDM2 and decreased levels of p53. Moreover, 27-OHC also enhanced physical interaction between p53 and MDM2. Furthermore, 27-OHC-induced proliferation was attenuated using either the p53 activator Tenovin-1 or the MDM2 inhibitor Nutlin-3 and Mdm2 siRNA. Taken together, our results indicate that 27-OHC may contribute to ER-positive breast cancer progression by disrupting constitutive p53 signaling in an MDM2-dependent manner.

Topics: Acetanilides; Breast Neoplasms; Cell Proliferation; Dose-Response Relationship, Drug; Female; Gene Expression Regulation, Neoplastic; Humans; Hydroxycholesterols; Imidazoles; MCF-7 Cells; Piperazines; Protein Binding; Proto-Oncogene Proteins c-mdm2; Receptors, Estrogen; RNA Interference; Selective Estrogen Receptor Modulators; Signal Transduction; Thiourea; Transcription, Genetic; Transfection; Tumor Suppressor Protein p53

Mouse double minute 2 (MDM2) is overexpressed in many malignant tumors, and MDM2 levels are associated with poor prognosis of several human cancers, including breast cancer. In the present study, we investigated the function of MDM2 in vascular endothelial growth factor (VEGF)-mediated tumor angiogenesis of breast cancer and the potential value of MDM2 as an anti-angiogenic therapy target for cancer therapy by inhibiting MDM2 with antisense oligonucleotides (ASO) or other antagonist nutlin-3.. Anti-MDM2 ASO and nutlin-3 were evaluated for their in vitro and in vivo anti-angiogenesis activities in different human breast cancer models with a different p53 status: MCF-7 cell line containing wild-type p53 and MDA-MB-468 cell line containing mutant p53. MCF-7 and MDA-MB-468 cells were incubated with different concentrations of ASO or nutlin-3 for various periods of time. VEGF gene and protein expression in tumor cells was measured by qPCR and Western blot. The level of VEGF protein secreted in the culture supernatant of treated cells was quantified by enzyme-linked immunosorbent assay (ELISA). Nude mouse xenograft models were further established to determine their effects on tumor growth and angiogenesis. Serum levels of VEGF were measured by ELISA. VEGF expression and microvessel density in tumor tissues were studied by immunohistochemistry. Both angiogenesis and tumor growth were digitally quantified.. In both MCF-7 and MDA-MB-468 cells, VEGF expression and secretion were reduced, resulting from specific inhibition of MDM2 expression by ASO. In vivo assay, after administration of ASO, VEGF production reduced and anti-angiogenesis activity occurred in nude mice bearing MCF-7 or MDA-MB-468 xenograft. However, in both models treated with nutlin-3, VEGF production was not changed and anti-angiogenesis activity was not observed.. In summary, the ASO construct targeting MDM2 specifically suppresses VEGF expression in vitro and VEGF-mediated tumor angiogenesis in vivo in breast cancer. Furthermore, the suppression of VEGF expression subsequent to inhibition of MDM2 in p53 mutant cells suggests that MDM2 has a regulatory role on VEGF expression through a p53-independent mechanism.

Topics: Animals; Breast Neoplasms; Cell Line, Tumor; Female; Heterografts; Humans; Imidazoles; Mice; Mice, Inbred BALB C; Mice, Nude; Neoplasm Transplantation; Neovascularization, Pathologic; Oligonucleotides, Antisense; Piperazines; Proto-Oncogene Proteins c-mdm2; Tumor Suppressor Protein p53; Vascular Endothelial Growth Factor A

Synthetic lethal interactions between poly (ADP-ribose) polymerase (PARP) and homologous recombination (HR) repair pathways have been exploited for the development of novel mono- and combination cancer therapies. The tumor suppressor p53 was demonstrated to exhibit indirect and direct regulatory activities in DNA repair, particularly in DNA double-strand break (DSB)-induced and replication-associated HR. In this study, we tested a potential influence of the p53 status on the response to PARP inhibition, which is known to cause replication stress. Silencing endogenous or inducibly expressing p53 we found a protective effect of p53 on PARP inhibitor (PARPi)-mediated cytotoxicities. This effect was specific for wild-type versus mutant p53 and observed in cancer but not in non-transformed cell lines. Enhanced cytotoxicities after treatment with the p53-inhibitory drug Pifithrinα further supported p53-mediated resistance to PARP inhibition. Surprisingly, we equally observed increased PARPi sensitivity in the presence of the p53-activating compound Nutlin-3. As a common denominator, both drug responses correlated with decreased HR activities: Pifithrinα downregulated spontaneous HR resulting in damage accumulation. Nutlin-3 induced a decrease of DSB-induced HR, which was accompanied by a severe drop in RAD51 protein levels. Thus, we revealed a novel link between PARPi responsiveness and p53-controlled HR activities. These data expand the concept of cell and stress type-dependent healer and killer functions of wild-type p53 in response to cancer therapeutic treatment. Our findings have implications for the individualized design of cancer therapies using PARPi and the potentially combined use of p53-modulatory drugs.

Topics: Benzothiazoles; Breast Neoplasms; Cell Line, Tumor; Enzyme Inhibitors; Female; Gene Knockdown Techniques; Genes, p53; Humans; Imidazoles; Isoquinolines; Molecular Weight; Piperazines; Poly(ADP-ribose) Polymerase Inhibitors; Poly(ADP-ribose) Polymerases; Recombinational DNA Repair; RNA, Small Interfering; Toluene

In the intrinsic pathway of apoptosis, cell-damaging signals promote the release of cytochrome c from mitochondria, triggering activation of the Apaf-1 and caspase-9 apoptosome. The ubiquitin E3 ligase MDM2 decreases the stability of the proapoptotic factor p53. We show that it also coordinated apoptotic events in a p53-independent manner by ubiquitylating the apoptosome activator CAS and the ubiquitin E3 ligase HUWE1. HUWE1 ubiquitylates the antiapoptotic factor Mcl-1, and we found that HUWE1 also ubiquitylated PP5 (protein phosphatase 5), which indirectly inhibited apoptosome activation. Breast cancers that are positive for the tyrosine receptor kinase HER2 (human epidermal growth factor receptor 2) tend to be highly aggressive. In HER2-positive breast cancer cells treated with the HER2 tyrosine kinase inhibitor lapatinib, MDM2 was degraded and HUWE1 was stabilized. In contrast, in breast cancer cells that acquired resistance to lapatinib, the abundance of MDM2 was not decreased and HUWE1 was degraded, which inhibited apoptosis, regardless of p53 status. MDM2 inhibition overcame lapatinib resistance in cells with either wild-type or mutant p53 and in xenograft models. These findings demonstrate broader, p53-independent roles for MDM2 and HUWE1 in apoptosis and specifically suggest the potential for therapy directed against MDM2 to overcome lapatinib resistance.

Topics: Animals; Apoptosis; Breast Neoplasms; Cell Line, Tumor; Drug Resistance, Neoplasm; Female; Humans; Imidazoles; Immunoblotting; Lapatinib; Mice; Mice, Nude; Myeloid Cell Leukemia Sequence 1 Protein; Nuclear Proteins; Phosphoprotein Phosphatases; Piperazines; Protein Kinase Inhibitors; Proto-Oncogene Proteins c-mdm2; Quinazolines; Receptor, ErbB-2; RNA Interference; Signal Transduction; Substrate Specificity; Tumor Suppressor Protein p53; Tumor Suppressor Proteins; Ubiquitin-Protein Ligases; Xenograft Model Antitumor Assays

The tumor suppressor gene p53 is often inactivated in breast cancer cells due to gene mutation or overexpression of its repressors (such as murine double minute 2 and murine double minute X). Inhibitors of murine double minute 2 (MDM2) and murine double minute X (MDMX) could lead to tumor suppression by restoration of p53 activity and such an approach is a promising strategy for future control of breast cancer. This study aimed to investigate the feasibility of the recombinant MDM2 and MDMX inhibitory protein in control of breast cancer in vitro. A cell-permeable dual-target MDM2/MDMX inhibitory protein was expressed in E. coli and incubated with p53 wild-type breast cancer cells. The data showed that this recombinant MDM2/MDMX inhibitory protein reduced the viability of MCF-7 and ZR-75-30 breast cancer cell lines and promoted cell cycle arrest and apoptosis by activation and stabilization of the p53 protein. Mechanistically, this MDM2/MDMX inhibitory protein increased the expression of p21, Bax and puma proteins, and inhibitory expression of MDM2 and MDMX proteins. This recombinant protein showed a better in vitro effect than that of nutlin-3α, a small molecule MDM2 inhibitor. The data further support the hypothesis that targeting of the p53 gene pathway could effectively control breast cancer.

Topics: Apoptosis; Apoptosis Regulatory Proteins; bcl-2-Associated X Protein; Breast Neoplasms; Cell Cycle Checkpoints; Cell Cycle Proteins; Cell Line, Tumor; Cell Survival; Cyclin-Dependent Kinase Inhibitor p21; Female; Humans; Imidazoles; MCF-7 Cells; Nuclear Proteins; Piperazines; Proto-Oncogene Proteins; Proto-Oncogene Proteins c-mdm2; Recombinant Fusion Proteins; Tumor Suppressor Protein p53

Links between a low vitamin D status and an increased risk of breast cancer have been observed in epidemiological studies. These links have been investigated in human tissue homogenates and cultured cell lines. We have used non-malignant, malignant and normal reduction mammoplasty breast tissues to investigate the biological and metabolic consequences of the application of vitamin D to intact ex vivo human breast tissue. Tissues were exposed to 1α,25(OH)(2)D(3) (1,25D; active metabolite) and 25(OH)D (25D; pre-metabolite). Changes in mRNA expression and protein expression after vitamin D exposure were analysed. Results indicate that while responses in normal and non-malignant breast tissues are similar between individuals, different tumour tissues are highly variable with regards to their gene expression and biological response. Collectively, malignant breast tissue responds well to active 1,25D, but not to the inactive pre-metabolite 25D. This may have consequences for the recommendation of vitamin D supplementation in breast cancer patients.

Topics: 25-Hydroxyvitamin D3 1-alpha-Hydroxylase; Breast Neoplasms; Calcifediol; Calcitriol; Cell Proliferation; Cyclin-Dependent Kinase Inhibitor p21; Female; Gene Expression; Humans; Imidazoles; Ki-67 Antigen; Piperazines; Proto-Oncogene Proteins c-mdm2; Receptors, Calcitriol; Steroid Hydroxylases; Tissue Culture Techniques; Up-Regulation; Vitamin D3 24-Hydroxylase

Tumor neovascularization (TNV) is a common pathologic basis for malignant growth and metastasis. However, the mechanism of TNV pathogenesis is not fully understood. Ras homolog gene family, member A (RhoA), a Rho guanosine triphosphatase (GTPase) family member, may be involved in a hypoxia-induced vascular endothelial growth factor (VEGF) pathway that regulates TNV angiogenesis through an unclear mechanism.. The regulation of RhoA on p53, the p53 binding protein homolog murine double minute 2 (MDM2), and VEGF was analyzed in hypoxic MCF-7 cells using Western blot analysis, real-time polymerase chain reaction (PCR) analysis, coimmunoprecipitation, and immunofluorescence staining assays. Changes in proliferation, invasion, migration, stress fiber formation, and tube formation were detected in an MCF-7 human umbilical vein endothelial cell (HUVEC) coculture system. Correlations of RhoA expression with MDM2, wild-type p53 (wt-p53), and VEGF expression in breast cancer tissues and relations between RhoA and breast cancer clinical features were analyzed by immunohistochemistry.. Activated RhoA down-regulated p53 protein, which increased VEGF expression in hypoxic MCF-7 cells; whereas p53 messenger RNA levels were not altered. In addition, the ubiquitin-mediated degradation of p53 was enhanced by active RhoA. RhoA and MDM2 colocalized in the cytoplasm of hypoxic MCF-7 cells and interacted with each other physically. Furthermore, nutlin-3, a specific MDM2 inhibitor, was capable of reducing activated RhoA-induced p53 protein stability and attenuating VEGF accumulation. In an MCF-7-HUVEC coculture system, nutlin-3 effectively inhibited HUVEC proliferation, invasion, migration, stress fiber formation, and tube formation mediated by activated RhoA under hypoxic conditions. Data from 129 clinical breast cancer specimens with wt-p53 revealed that high RhoA expression was correlated with high MDM2 expression, low wt-p53 expression, and high VEGF expression.. The current data suggested that activated RhoA promotes VEGF expression and hypoxia-induced angiogenesis through the up-regulation of MDM2 to decrease p53 stability.

Topics: Animals; Breast Neoplasms; Cell Hypoxia; Cell Line; Cell Line, Tumor; Cell Movement; Coculture Techniques; Cytoprotection; Female; Gene Expression Regulation, Neoplastic; Genes, ras; Human Umbilical Vein Endothelial Cells; Humans; Imidazoles; Mice; Middle Aged; Neovascularization, Pathologic; Piperazines; Proto-Oncogene Proteins c-mdm2; rhoA GTP-Binding Protein; Ubiquitin; Vascular Endothelial Growth Factor A

The objective of the present study is to prepare and characterize nutlin-3a loaded polymeric poly(lactide-co-glycolide) nanoparticles (NPs) surface functionalized with transferrin ligand, to deliver the encapsulated drug in a targeted manner to its site of action and to evaluate the efficacy of the nanoformulation in terms of its cellular uptake, cell cytotoxicity, cell cycle arrest, apoptosis and activation of p53 pathway at molecular level in MCF-7 breast cancer cell line.. Nutlin-3a loaded poly(lactide-co-glycolide) NPs were prepared following the single oil-in-water emulsion method. Physicochemical characterization of the formulation included size and surface charge measurement, transmission electron microscopy characterization, study of surface morphology using scanning electron microscopy, Fourier-transform infrared spectral analysis and in vitro release kinetics studies. Furthermore, targeting ability of the conjugated system was assessed by cellular uptake and cell cytotoxicity studies in an in vitro cell model. Molecular basis of nutlin-3a-mediated p53 activation pathway was investigated by western blot analysis. Inhibition of cell cycle progression and apoptosis was evaluated by flow cytometry.. Physiochemical characterization of the formulations revealed that nutlin-3a was efficiently encapsulated in the nanoparticulate system, reaching an encapsulation efficiency of approximately 80% with size of approximately 220 nm and negative zeta potential of approximately -10.4 mV. Higher cellular uptake efficiency of the conjugated system proved the effectiveness of targeted therapy. IC(50) values, as determined by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium assay, showed superior antiproliferative activity of transferrin-conjugated NPs over unconjugated NPs and native nutlin-3a, owing to enhanced cellular uptake by cancer cells. At the molecular level the conjugated system showed enhanced activation of p53 pathway in comparison to native drug as evident from western blot analysis. Augmented cell cycle arrest and apoptosis was exhibited by the conjugated system. Thus, our results suggest that transferrin-conjugated nutlin-3a loaded NPs could be a potential drug carrier system for targeted delivery of potent anticancer drug nutlin-3a for breast cancer therapy.

Topics: Apoptosis; Breast Neoplasms; Cell Line, Tumor; Cell Survival; Drug Delivery Systems; Female; Humans; Imidazoles; Nanoparticles; Piperazines; Proto-Oncogene Proteins c-mdm2; Tumor Suppressor Protein p53

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

The tumor suppressor p53 is often inactivated in breast cancer cells because the overexpression of its repressors (e.g., MDM2 and MDMX). Restoration of p53 activity by small molecules through counteracting p53 repressors can lead to in vivo tumor regression and is therefore considered a promising strategy for treatments of cancer. Recent efforts in high-throughput drug screening and rational drug design have identified several structurally diverse small-molecule p53 activators, including a pseudourea derivative XI-011 (NSC146109). This small molecule strongly activates p53 while selectively inhibiting growth of transformed cells without inducing genotoxicity, indicating its potential as a drug lead for p53-targeted therapy. However, the mechanism(s) by which XI-011 activates p53 and the effects of XI-011 on growth of breast cancer cells are currently unknown. Here, we report that XI-011 promoted breast cancer cells to undergo apoptosis through activating p53 and inducing expression of proapoptotic genes. Importantly, we found that activation of p53 by this small molecule was achieved through a novel mechanism, that is, inhibition of MDMX expression. XI-011 repressed the MDMX promoter, resulting in down-regulation of MDMX messenger RNA level in MCF-7 cells. In line with these results, XI-011 decreased the viability of breast cancer cells expressing low levels of MDMX in a less-efficient manner. Interestingly, XI-011 acted additively with the MDM2 antagonist Nutlin-3a to inhibit growth of breast cancer cells. We conclude that XI-011 belongs to a novel class of small-molecule p53 activators that target MDMX and could be of value in treating breast cancer.

Topics: Anthracenes; Antineoplastic Agents; Antineoplastic Combined Chemotherapy Protocols; Apoptosis; Breast Neoplasms; Carcinoma; Cell Cycle Proteins; Cell Line, Tumor; Down-Regulation; Drug Evaluation, Preclinical; Drug Synergism; Female; Gene Expression Regulation, Neoplastic; Humans; Imidazoles; Models, Biological; Molecular Weight; Nuclear Proteins; Piperazines; Proto-Oncogene Proteins; Thiourea; Tumor Cells, Cultured; Tumor Suppressor Protein p53

The E3 ubiquitin ligase human murine double minute (HDM2) is overexpressed in 40%-80% of late-stage metastatic cancers in the absence of gene amplification. Hdm2 regulates p53 stability via ubiquitination and has also been implicated in altering the sensitivity of cells to TGF-beta1. Whether TGF-beta1 signaling induces Hdm2 expression leading to HDM2-mediated destabilization of p53 has not been investigated. In this study, we report that TGF-beta1-activated SMA- and MAD3 (Smad3/4) transcription factors specifically bound to the second promoter region of HDM2, leading to increased HDM2 protein expression and destabilization of p53 in human cancer cell lines. Additionally, TGF-beta1 expression led to Smad3 activation and murine double minute 2 (Mdm2) expression in murine mammary epithelial cells during epithelial-to-mesenchymal transition (EMT). Furthermore, histological analyses of human breast cancer samples demonstrated that approximately 65% of late-stage carcinomas were positive for activated Smad3 and HDM2, indicating a strong correlation between TGF-beta1-mediated induction of HDM2 and late-stage tumor progression. Identification of Hdm2 as a downstream target of TGF-beta1 represents a critical prosurvival mechanism in cancer progression and provides another point for therapeutic intervention in late-stage cancer.

Topics: Apoptosis; Breast Neoplasms; Female; Gene Expression Regulation, Neoplastic; HCT116 Cells; Humans; Imidazoles; Neoplasm Staging; Piperazines; Promoter Regions, Genetic; Proto-Oncogene Proteins c-mdm2; Smad3 Protein; Transforming Growth Factor beta1; Tumor Suppressor Protein p53

The forkhead transcription factor, Foxp3, is thought to act as a master regulator that controls (suppresses) expression of the breast cancer oncogenes, SKP2 and HER-2/ErbB2. However, the mechanisms that regulate Foxp3 expression and thereby modulate tumor development remain largely unexplored. Here, we demonstrate that Foxp3 up-regulation requires p53 function, showing that Foxp3 expression is directly regulated by p53 upon DNA damage responses in human breast and colon carcinoma cells. Treatment with the genotoxic agents, doxorubicin or etoposide, induced Foxp3 expression in p53-positive carcinoma cells, but not in cells lacking p53 function. Furthermore, knock down of endogenous wild-type p53 using RNA interference abrogated Foxp3 induction by genotoxic agents, and exogenous expression of p53 in cells lacking p53 restored the responsiveness of Foxp3 to DNA-damaging stresses. In addition, Foxp3 knock down blunted the p53-mediated growth inhibitory response to DNA-damaging agents. These results suggest that induction of Foxp3 in the context of tumor suppression is regulated in a p53-dependent manner and implicate Foxp3 as a key determinant of cell fate in p53-dependent DNA damage responses.

Topics: Antibiotics, Antineoplastic; Antineoplastic Agents, Phytogenic; Breast Neoplasms; Cell Death; Cell Division; Colonic Neoplasms; DNA Damage; Doxorubicin; Etoposide; Female; Forkhead Transcription Factors; Gene Expression Regulation, Neoplastic; HCT116 Cells; Humans; Imidazoles; Phosphorylation; Piperazines; Proto-Oncogene Proteins c-mdm2; Tumor Suppressor Protein p53

The p53 tumor suppressor protein is frequently mutated in human tumors. It is thought that the p53 pathway is indirectly impaired in the remaining tumors, for example by overexpression of its important regulators Mdm2 and Mdm4, making them attractive targets for the development of anti-cancer agents. Recent studies have suggested that Mdm4 levels determine the sensitivity of tumor cells for anti-cancer therapy. To investigate this possibility, we studied the drug sensitivity of several breast cancer cell lines containing wild-type p53, but expressing different Mdm4 levels. We show that endogenous Mdm4 levels can affect the sensitivity of breast cancer cells to anti-cancer agents, but in a cell line-dependent manner and depending on an intact apoptotic response. Furthermore, treatment with the non-genotoxic agent Nutlin-3 sensitizes cells for doxorubicin, showing that activation of p53 by targeting its regulators is an efficient strategy to decrease cell viability of breast cancer cells. These results confirm a function of Mdm4 in determining the efficacy of chemotherapeutic agents to induce apoptosis of cancer cells in a p53-dependent manner, although additional undetermined factors also influence the drug response. Targeting Mdm4 to sensitize tumor cells for chemotherapeutic drugs might be a strategy to effectively treat tumors harboring wild-type p53.

Topics: Apoptosis; Breast Neoplasms; Cell Cycle Proteins; Cell Line, Tumor; Doxorubicin; Drug Resistance, Neoplasm; Female; Humans; Imidazoles; Nuclear Proteins; Piperazines; Proto-Oncogene Proteins; Tumor Suppressor Protein p53

Topics: Animals; Breast Neoplasms; Cell Cycle; Cellular Senescence; Female; Humans; Imidazoles; Piperazines; TOR Serine-Threonine Kinases; Tumor Suppressor Protein p53

p53 is inactivated in many human malignancies through missense mutations or overexpression of the human homologue of Mdm2 (Hdm2), an E3 ubiquitin ligase that ubiquitinates p53, thereby promoting its proteasomal degradation. The cis-imidazoline nutlin-3 can disrupt the p53-Hdm2 interaction and activate p53, inducing apoptosis in vitro in many malignancies, including multiple myeloma (MM).. We hypothesized that suppression of Hdm2-mediated p53 ubiquitination may augment sequelae of p53 accumulation caused by proteasomal inhibition. We compared the response of MM cells versus several epithelial cancer models to the proteasome inhibitor bortezomib in combination with nutlin-3.. The combination of sublethal concentrations of bortezomib plus nutlin-3 induced additive cytotoxicity against bortezomib-sensitive MM cell lines. Importantly, however, in breast, prostate, colon, and thyroid (papillary, follicular, anaplastic, and medullary) carcinoma cell lines, this combination triggered synergistic cytotoxicity, and increased expression of p53, p21, Hdm2, Bax, Noxa, PUMA, and cleavage of caspase-3 and poly ADP ribose polymerase. Coculture with bone marrow stromal cells attenuated MM cell sensitivity to nutlin-3 monotherapy and was associated with evidence of suppression of p53 activity in MM cells, whereas combined bortezomib-nutlin-3 treatment maintained cytotoxicity even in the presence of bone marrow stromal cells.. This differential response of MM versus epithelial carcinomas to combination of nutlin-3 with bortezomib sheds new light on the role of p53 in bortezomib-induced apoptosis. Concurrent Hdm2 inhibition with bortezomib may extend the spectrum of bortezomib applications to malignancies with currently limited sensitivity to single-agent bortezomib or, in the future, to MM patients with decreased clinical responsiveness to bortezomib-based therapy.

Topics: Antineoplastic Agents; Apoptosis; Boronic Acids; Bortezomib; Breast Neoplasms; Carcinoma; Cell Line, Tumor; Cell Survival; Humans; Imidazoles; Multiple Myeloma; Mutation, Missense; Neoplasms, Glandular and Epithelial; Piperazines; Proteasome Endopeptidase Complex; Proteasome Inhibitors; Proto-Oncogene Proteins c-mdm2; Pyrazines; Tumor Suppressor Protein p53

Recent studies have shown that activation of cell cycle checkpoints can protect normal proliferating cells from mitotic inhibitors by preventing their entry into mitosis. These studies have used genotoxic agents that act, at least in part, by activation of the p53 pathway. However, genotoxic drugs are known also to have p53-independent activities and could affect the sensitivity of tumor cells to antimitotic agents. Recently, we have developed the first potent and selective small-molecule inhibitors of the p53-MDM2 interaction, the nutlins, which activate the p53 pathway only in cells with wild-type but not mutant p53. Using these compounds, we show that p53 activation leads to G1 and G2 phase arrest and can protect cells from mitotic block and apoptosis caused by paclitaxel. Pretreatment of HCT116 and RKO colon cancer cells (wild-type p53) or primary human fibroblasts (1043SK) with nutlins for 24 hours followed by incubation with paclitaxel for additional 48 hours did not increase significantly their mitotic index and protected the cells from the cytotoxicity of paclitaxel. Cancer cells with mutant p53 (MDA-MB-435) responded to the same treatment with mitotic arrest and massive apoptosis. These results have two major implications for cancer therapy. First, p53-activating therapies may have antagonistic effect when combined with mitotic poisons. Second, pretreatment with MDM2 antagonists before chemotherapy of tumors with mutant p53 may offer a partial protection to proliferating normal tissues.

Topics: Antineoplastic Agents, Phytogenic; Apoptosis; Breast Neoplasms; Cell Proliferation; Colonic Neoplasms; Female; G1 Phase; G2 Phase; Humans; Imidazoles; Mitosis; Mutation; Nuclear Proteins; Paclitaxel; Piperazines; Proto-Oncogene Proteins; Proto-Oncogene Proteins c-mdm2; Tumor Cells, Cultured; Tumor Suppressor Protein p53