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Glioblastoma multiforme (GBM), also known as grade IV astrocytoma, represents the most aggressive primary brain tumor. The complex genetic heterogeneity, the acquired drug resistance, and the presence of the blood-brain barrier (BBB) limit the efficacy of the current therapies, with effectiveness demonstrated only in a small subset of patients. To overcome these issues, here we propose an anticancer approach based on ultrasound-responsive drug-loaded organic piezoelectric nanoparticles. This anticancer nanoplatform consists of nutlin-3a-loaded ApoE-functionalized P(VDF-TrFE) nanoparticles, that can be remotely activated with ultrasound-based mechanical stimulations to induce drug release and to locally deliver anticancer electric cues. The combination of chemotherapy treatment with chronic piezoelectric stimulation resulted in activation of cell apoptosis and anti-proliferation pathways, induction of cell necrosis, inhibition of cancer migration, and reduction of cell invasiveness in drug-resistant GBM cells. Obtained results pave the way for the use of innovative multifunctional nanomaterials in less invasive and more focused anticancer treatments, able to reduce drug resistance in GBM. Piezoelectric hybrid lipid-polymeric nanoparticles, efficiently encapsulating a non-genotoxic drug (nutlin-3a) and functionalized with a peptide (ApoE) that enhances their passage through the BBB, are proposed. Upon ultrasound stimulation, nanovectors resulted able to reduce cell migration, actin polymerization, and invasion ability of glioma cells, while fostering apoptotic and necrotic events. This wireless activation of anticancer action paves the way to a less invasive, more focused and efficient therapeutic strategy. [Display omitted] [ABSTRACT FROM AUTHOR]

Colorectal cancer (CRC) is the third most common and the second deadliest type of cancer worldwide, urging the development of more comprehensive models and of more efficient treatments. Although the combination of nanotechnology with chemo- and immuno-therapy has represented a promising treatment approach, its translation to the clinic has been hampered by the absence of cellular models that can provide reliable and predictive knowledge about the in vivo efficiency of the formulation. Herein, a 3D model based on CRC multicellular tumor spheroids (MCTS) model was developed by combining epithelial colon cancer cells (HCT116), human intestinal fibroblasts and monocytes. The developed MCTS 3D model mimicked several tumor features with cells undergoing spatial organization and producing extracellular matrix, forming a mass of tissue with a necrotic core. Furthermore, monocytes were differentiated into macrophages with an anti-inflammatory, pro-tumor M2-like phenotype. For a combined chemoimmunotherapy effect, spermine-modified acetalated dextran nanoparticles (NPs) loaded with the chemotherapeutic Nutlin-3a (Nut3a) and granulocyte-macrophage colony-stimulating factor (GM-CSF) were produced and tested in 2D cultures and in the MCTS 3D model. NPs were successfully taken-up by the cells in 2D, but in a significant less extent in the 3D model. However, these NPs were able to induce an anti-proliferative effect both in the 2D and in the 3D models. Moreover, Nut3a was able to partially shift the polarization of the macrophages present in the MCTS 3D model towards an anti-tumor M1-like phenotype. Overall, the developed MCTS 3D model showed to recapitulate key features of tumors, while representing a valuable model to assess the effect of combinatorial nano-therapeutic strategies in CRC. In addition, the developed NPs could represent a promising approach for CRC treatment. Unlabelled Image [ABSTRACT FROM AUTHOR]

Mutations at the TP53 gene are readily detected (approximately 50–75%) in pancreatic ductal adenocarcinoma (PDAC) patients. TP53 was previously thought to be a difficult target as it is often mutated, deleted or inactivated on both chromosomes in certain cancers. In the following study, the effects of restoration of wild-type (WT) TP53 activity on the sensitivities of MIA-PaCa-2 pancreatic cancer cells to the MDM2 inhibitor nutlin-3a in combination with chemotherapy, targeted therapy, as well as, nutraceuticals were examined. Upon introduction of the WT- TP53 gene into MIA-PaCa-2 cells, which contain a TP53 gain of function (GOF) mutation, the sensitivity to the MDM2 inhibitor increased. However, effects of nutlin-3a were also observed in MIA-PaCa-2 cells lacking WT-TP53, as upon co-treatment with nutlin-3a, the sensitivity to certain inhibitors, chemotherapeutic drugs and nutraceuticals increased. Interestingly, co-treatment with nutlin-3a and certain chemotherapeutic drug such as irinotecan and oxaliplatin resulted in antagonistic effects in cells both lacking and containing WT-TP53 activity. These studies indicate the sensitizing abilities that WT- TP53 activity can have in PDAC cells which normally lack WT- TP53 , as well as, the effects that the MDM2 inhibitor nutlin-3a can have in both cells containing and lacking WT-TP53 to various therapeutic agents. [ABSTRACT FROM AUTHOR]

TREM2 mutations evoke neurodegenerative disorders, and recently genetic variants of this gene were correlated to increased risk of Alzheimer's disease. The signaling cascade originating from the TREM2 membrane receptor includes its binding partner TYROBP, BLNK adapter protein, and SYK kinase, which can be activated by p53. Moreover, in silico identification of a putative p53 response element (RE) at the TREM2 promoter led us to hypothesize that TREM2 and other pathway elements may be regulated in p53-dependent manner. To stimulate p53 in synergistic fashion, we exposed A549 lung cancer cells to actinomycin D and nutlin-3a (A + N). In these cells, exposure to A + N triggered expression of TREM2, TYROBP, SYK and BLNK in p53-dependent manner. TREM2 was also activated by A + N in U-2 OS osteosarcoma and A375 melanoma cell lines. Interestingly, nutlin-3a, a specific activator of p53, acting alone stimulated TREM2 in U-2 OS cells. Using in vitro mutagenesis, chromatin immunoprecipitation, and luciferase reporter assays, we confirmed the presence of the p53 RE in TREM2 promoter. Furthermore, activation of TREM2 and TYROBP by p53 was strongly inhibited by CHIR-98014, a potent and specific inhibitor of glycogen synthase kinase-3 (GSK-3). We conclude that TREM2 is a direct p53-target gene, and that activation of TREM2 by A + N or nutlin-3a may be critically dependent on GSK-3 function. [ABSTRACT FROM AUTHOR]

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. From the Clinical Editor 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. [ABSTRACT FROM AUTHOR]

Acute myeloid leukemia (AML) is a heterogeneous disease with variable patient responses to therapy. Selinexor, an inhibitor of nuclear export, has shown promising clinical activity for AML. To identify the molecular context for monotherapy sensitivity as well as rational drug combinations, we profile selinexor signaling responses using phosphoproteomics in primary AML patient samples and cell lines. Functional phosphosite scoring reveals that p53 function is required for selinexor sensitivity consistent with enhanced efficacy of selinexor in combination with the MDM2 inhibitor nutlin-3a. Moreover, combining selinexor with the AKT inhibitor MK-2206 overcomes dysregulated AKT-FOXO3 signaling in resistant cells, resulting in synergistic anti-proliferative effects. Using high-throughput spatial proteomics to profile subcellular compartments, we measure global proteome and phospho-proteome dynamics, providing direct evidence of nuclear translocation of FOXO3 upon combination treatment. Our data demonstrate the potential of phosphoproteomics and functional phosphorylation site scoring to successfully pinpoint key targetable signaling hubs for rational drug combinations. [Display omitted] • Phosphoproteomics with functional scoring uncovers context for selinexor sensitivity • Functional p53 correlates with selinexor sensitivity, which is enhanced by nutlin-3a • Dysregulated AKT-FOXO3 drives selinexor resistance, which is overcome with MK-2206 • Spatial proteomics reveals selinexor-induced nucleocytoplasmic protein shuttling Emdal et al. combine phosphoproteomics of samples from patients with AML and functional phosphosite scoring to uncover clinically actionable molecular context for selinexor efficacy. Sensitivity to selinexor correlates with functional p53 and is enhanced with nutlin-3a, while resistance is associated with dysregulated AKT-FOXO3 signaling and overcome by combining with MK-2206. [ABSTRACT FROM AUTHOR]

Highlights: [•] Inhibition of α5β1 integrin expression or functions alters HCT116 cell survival. [•] Nutlin-3a affects α5 integrin expression through p53 activation. [•] A negative crosstalk between α5β1 integrin and p53 wt has been identified. [•] Reactivation of p53 above a threshold is of interest as a therapeutic option for colon tumors overexpressing α5 integrin. [Copyright &y& Elsevier]

Abstract: Breast cancer resistance protein (BCRP; ABCG2), a clinical marker for identifying the side population (SP) cancer stem cell subgroup, affects intestinal absorption, brain penetration, hepatobiliary excretion, and multidrug resistance of many anti-cancer drugs. Nutlin-3a is currently under pre-clinical investigation in a variety of solid tumor and leukemia models as a p53 reactivation agent, and has been recently demonstrated to also have p53 independent actions in cancer cells. In the present study, we first report that nutlin-3a can inhibit the efflux function of BCRP. We observed that although the nutlin-3a IC50 did not differ between BCRP over-expressing and vector control cells, nutlin-3a treatment significantly potentiated the cells to treatment with the BCRP substrate mitoxantrone. Combination index calculations suggested synergism between nutlin-3a and mitoxantrone in cell lines over-expressing BCRP. Upon further investigation, it was confirmed that nutlin-3a increased the intracellular accumulation of BCRP substrates such as mitoxantrone and Hoechst 33342 in cells expressing functional BCRP without altering the expression level or localization of BCRP. Interestingly, nutlin-3b, considered virtually “inactive” in disrupting the MDM2/p53 interaction, reversed Hoechst 33342 efflux with the same potency as nutlin-3a. Intracellular accumulation and bi-directional transport studies using MDCKII cells suggested that nutlin-3a is not a substrate of BCRP. Additionally, an ATPase assay using Sf9 insect cell membranes over-expressing wild-type BCRP indicated that nutlin-3a inhibits BCRP ATPase activity in a dose-dependent fashion. In conclusion, our studies demonstrate that nutlin-3a inhibits BCRP efflux function, which consequently reverses BCRP-related drug resistance. [Copyright &y& Elsevier]

Abstract: 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. [ABSTRACT FROM AUTHOR]

Abstract: A sensitive and precise LC–ESI-MS/MS method for determination of nutlin-3a in murine plasma using ketoconazole as an internal standard was developed and validated. Plasma nutlin-3a samples were prepared by either a simple protein precipitation (PP) for the high concentration range (10–20,000ng/mL) or by liquid–liquid extraction (LLE) for the low concentration range (0.25–300ng/mL). Nutlin-3a and ketoconazole were separated on a modified C18 analytical column (4μm, 75mm×2mm) with an isocratic mobile phase (acetonitrile/5mM HCOONH4 =70/30, v/v). The retention times of nutlin-3a and ketoconazole were 1.14 and 1.45min. Detection was achieved by a tandem MS system, monitoring m/z 582/99 and m/z 532/82 for nutlin-3a and ketoconazole, respectively. The PP method was linear in a range of 10–20,000ng/mL (R 2 ≥0.993) and the LLE method was linear in a range of 0.25–300ng/mL (R 2 ≥0.992). The mean recoveries for PP and LLE were 24% and 78%, respectively. Within-day and between-day precisions were ≤4.5% for PP and were ≤4.9% for LLE. Within-day and between-day accuracies (% error) ranged from 4.8 to −7.9 for PP, and from −0.2 to −8.4 for LLE. The two extraction methods produced equivalent results, allowing use of both within the same study. This method has been applied to the measurement of nutlin-3a concentrations in murine plasma samples obtained from a preclinical pharmacokinetic study. [Copyright &y& Elsevier]

DNA-binding transcription factors (TFs) remain challenging to target with molecular probes. Many TFs function in part through interaction with Mediator, a 26-subunit complex that controls RNA polymerase II activity genome-wide. We sought to block p53 function by disrupting the p53-Mediator interaction. Through rational design and activity-based screening, we characterize a stapled peptide, with functional mimics of both p53 activation domains, that blocks p53-Mediator binding and selectively inhibits p53-dependent transcription in human cells; importantly, this "bivalent" peptide has negligible impact, genome-wide, on non-p53 target genes. Our proof-of-concept strategy circumvents the TF entirely and targets the TF-Mediator interface instead, with desired functional outcomes (i.e., selective inhibition of p53 activation). Furthermore, these results demonstrate that TF activation domains represent viable starting points for Mediator-targeting molecular probes, as an alternative to large compound libraries. Different TFs bind Mediator through different subunits, suggesting this strategy could be broadly applied to selectively alter gene expression programs. [Display omitted] • Proteomics identifies Mediator (MED) as "top hit" interactor with p53AD • Stapled peptide inhibits p53-MED binding and p53 transcription; other TFs not affected • TF activation domains viable starting points for design of Mediator-targeting probes • TF function can be controlled by avoiding TF entirely and targeting Mediator instead Allen et al. combine chemical synthesis and biochemistry to develop a Mediator-targeting "bivalent" peptide that blocks p53-Mediator binding and selectively inhibits p53 target gene expression in human cells. This proof-of-concept study shows that p53 activity can be blocked by molecules targeting p53-Mediator binding, circumventing p53 itself. [ABSTRACT FROM AUTHOR]

Approaches to improve pancreatic cancer therapy are essential as this disease has a very bleak outcome. Approximately 80% of pancreatic cancers are pancreatic ductal adenocarcinomas (PDAC). A key regulatory gene frequently mutated (∼75%) in PDAC is the TP53 tumor suppressor gene which controls the transcription of multiple genes involved in cell cycle progression, apoptosis, cancer progression and other growth regulatory processes. The mouse double minute 2 homolog (MDM2) gene product is a nuclear-localized E3 ubiquitin ligase and negatively regulates the TP53 protein which results in its proteasomal degradation. Various MDM2 inhibitors have been isolated and examined in clinical trials, especially in patients with hematological malignancies. Nutlin-3a is one of the first MDM2 inhibitors isolated. Berberine (BBR) is a natural product found in many fruits and berries and used in traditional medicine for centuries. It has many biological effects, and some are anti-proliferative in nature. BBR may activate the expression of TP53 and inhibit cell cycle progression as well as other events important in cell growth. To understand more about the potential of compounds like BBR and chemical modified BBRs (NAX compounds) to sensitize PDAC cells to MDM2 inhibitors, we introduced either WT-TP53 or the pLXSN empty vector control into two PDAC cell lines, one lacking expression of TP53 (PANC-28) and one with gain-of-function mutant TP53 on both alleles (MIA-PaCa-2). Our results indicate that nutlin-3a was able to increase the sensitivity to BBR and certain NAX compounds. The effects of nutlin-3a were usually more substantial in those cells containing an introduced WT TP53 gene. These results highlight the importance of knowledge of the type of TP53 mutation that is present in cancer patients before the administration of drugs which function by stabilization of the TP53 protein. [ABSTRACT FROM AUTHOR]

The problem of chemoresistance development is an inescapable flipside of modern oncotherapy, in particular for сolorectal cancer patients. The search for or development of drugs effective against resistant tumors involves the use of model resistant cell lines in vitro. To obtain such lines, we reproduced the development of chemoresistance of human colon adenocarcinoma cells under the treatment with drugs of different mechanisms, a cytostatic (paclitaxel) and a targeted agent (Nutlin-3a, an inhibitor of p53-Mdm2 protein-protein interaction). In each case, we gradually increased the content of the substance in the medium, starting from effective concentrations that do not cause total cell death. When studying the lines resistant to the corresponding drug, we noted a reduced sensitivity to the drug of another mechanism of action. Analysis of the original and resistant lines showed that the cells use the universal efflux defense mechanism. The observed effect can be partially neutralized using inhibitors of the ABC transport proteins, including P-glycoprotein, known for its oncoprotective function. The role of the latter was confirmed by real-time RT-PCR and Western blotting. [Display omitted] • Both Mdm2 inhibitor and cytostatic agent can provoke the development of chemoresistance in tumor cells. • Each of the drugs causes a decrease in the sensitivity of cells to the agent with another mechanism of action. • Efflux is significantly enhanced in resistant cells and is determined by increased P-glycoprotein expression. [ABSTRACT FROM AUTHOR]

Nutlin-3a is a p53 activator and potential cyclotherapy approach that may also mitigate side effects of chemotherapeutic drugs in the treatment of colorectal cancer. We investigated cell proliferation in a panel of colorectal cancer (CRC) cell lines with wild-type or mutant p53, as well as a non-tumorigenic fetal intestinal cell line following Nutlin-3a treatment (10 μM). We then assessed apoptosis at 24 and 48 h following administration of the active irinotecan metabolite, SN-38 (0.001 μM - 1 μM), alone or following pre-treatment with Nutlin-3a (10 μM). Nutlin-3a treatment (10 μM) significantly reduced proliferation in wild-type p53 expressing cell lines (FHS 74 and HCT116+/+) at 72 and 96 h, but was without effect in cell lines with mutated or deleted p53 (Caco-2, SW480, and HCT 116−/−). SN-38 treatment induced significant apoptosis in all cell lines after 48 h. Nutlin-3a unexpectedly increased cell death in the p53 wild-type CRC cell line, HCT116+/+, while Nutlin-3a pre-treatment provided protection from SN-38 in the p53 wild-type normal cell line, FHs 74. These results demonstrate Nutlin-3a's selective growth-arresting efficacy in p53 wild-type non-malignant intestinal cell lines, enabling the selective targeting of malignant cells with chemotherapy drugs. These studies highlight the potential of Nutlin-3a to minimise intestinal mucosal damage following chemotherapy. • p53 inhibitor Nutlin-3a may protect intestinal epithelial cells during chemotherapy. • Nutlin-3a reduced proliferation in p53 wild-type but not p53 aberrant cells. • SN-38 induced more potent cytotoxicity in colorectal cancer cells than FHs-74 cells. • Nutlin-3a protected FHs-74 cells, but not WT p53 HCT cells from SN-38 toxicity. • Nutlin-3a may provide protection from chemotherapy-related intestinal toxicity. [ABSTRACT FROM AUTHOR]