Anticancer Activity of Aqueous Extracts from Asparagus officinalis L. Byproduct on Breast Cancer Cells.

Cultivation of asparagus (Asparagus officinalis L.; Asp) for food and medicinal use has taken place since the early Roman Empire. Today, Asp represents a worldwide diffuse perennial crop. Lower portions of the spears represent a food industry waste product that can be used to extract bioactive molecules. In this study, aqueous extracts derived from the non-edible portion of the plant (hard stem) were prepared and characterized for chemical content. Furthermore, the biocompatibility and bioactivity of Asp aqueous extracts were assessed in vitro on normal fibroblasts and on breast cancer cell lines. Results showed no interference with fibroblast viability, while a remarkable cytostatic concentration-dependent activity, with significant G1/S cell cycle arrest, was specifically observed in breast cancer cells without apoptosis induction. Asp extracts were also shown to significantly inhibit cell migration. Further analyses showed that Asp extracts were characterized by specific pro-oxidant activity against tumoral cells, and, importantly, that their combination with menadione resulted in a significant enhancement of oxidants production with respect to menadione alone in breast cancer cells but not in normal cells. This selectivity of action on tumoral cells, together with the easiness of their preparation, makes the aqueous Asp extracts very attractive for further investigation in breast cancer research, particularly to investigate their role as possible co-adjuvant agents of clinical drug therapies.

TRAIL, OPG, and TWEAK in kidney disease: biomarkers or therapeutic targets?

Ligands and receptors of the tumor necrosis factor (TNF) superfamily regulate immune responses and homeostatic functions with potential diagnostic and therapeutic implications. Kidney disease represents a global public health problem, whose prevalence is rising worldwide, due to the aging of the population and the increasing prevalence of diabetes, hypertension, obesity, and immune disorders. In addition, chronic kidney disease is an independent risk factor for the development of cardiovascular disease, which further increases kidney-related morbidity and mortality. Recently, it has been shown that some TNF superfamily members are actively implicated in renal pathophysiology. These members include TNF-related apoptosis-inducing ligand (TRAIL), its decoy receptor osteoprotegerin (OPG), and TNF-like weaker inducer of apoptosis (TWEAK). All of them have shown the ability to activate crucial pathways involved in kidney disease development and progression (e.g. canonical and non-canonical pathways of the transcription factor nuclear factor-kappa B), as well as the ability to regulate cell proliferation, differentiation, apoptosis, necrosis, inflammation, angiogenesis, and fibrosis with double-edged effects depending on the type and stage of kidney injury. Here we will review the actions of TRAIL, OPG, and TWEAK on diabetic and non-diabetic kidney disease, in order to provide insights into their full clinical potential as biomarkers and/or therapeutic options against kidney disease.

Changes in expression profiles of internal jugular vein wall and plasma protein levels in multiple sclerosis.

BACKGROUND: Multiple sclerosis (MS) is an inflammatory, demyelinating and degenerative disorder of the central nervous system (CNS). Several observations support interactions between vascular and neurodegenerative mechanisms in multiple sclerosis (MS). To investigate the contribution of the extracranial venous compartment, we analysed expression profiles of internal jugular vein (IJV), which drains blood from CNS, and related plasma protein levels. METHODS: We studied a group of MS patients (n = 19), screened by echo-color Doppler and magnetic resonance venography, who underwent surgical reconstruction of IJV for chronic cerebrospinal venous insufficiency (CCSVI). Microarray-based transcriptome analysis was conducted on specimens of IJV wall from MS patients and from subjects undergoing carotid endarterectomy, as controls. Protein levels were determined by multiplex assay in: i) jugular and peripheral plasma from 17 MS/CCSVI patients; ii) peripheral plasma from 60 progressive MS patients, after repeated sampling and iii) healthy individuals. RESULTS: Of the differentially expressed genes (≥ 2 fold-change, multiple testing correction, P < 0.05), the immune-related CD86 (8.5 fold-change, P = 0.002) emerged among the up regulated genes (N = 409). Several genes encoding HOX transcription factors and histones potentially regulated by blood flow, were overexpressed. Smooth muscle contraction and cell adhesion processes emerged among down regulated genes (N = 515), including the neuronal cell adhesion L1CAM as top scorer (5 fold-change, P = 5 × 10- 4). Repeated measurements in jugular/peripheral plasma and overtime in peripheral plasma showed conserved individual plasma patterns for immune-inflammatory (CCL13, CCL18) and adhesion (NCAM1, VAP1, SELL) proteins, despite significant variations overtime (SELL P < 0.0001). Both age and MS disease phenotypes were determinants of VAP1 plasma levels. Data supported cerebral related-mechanisms regulating ANGPT1 levels, which were remarkably lower in jugular plasma and correlated in repeated assays but not between jugular/peripheral compartments. CONCLUSIONS: This study provides for the first time expression patterns of the IJV wall, suggesting signatures of altered vascular mRNA profiles in MS disease also independently from CCSVI. The combined transcriptome-protein analysis provides intriguing links between IJV wall transcript alteration and plasma protein expression, thus highlighting proteins of interest for MS pathophysiology.

Clinical perspectives of TRAIL: insights into central nervous system disorders.

The TNF-related apoptosis inducing ligand TRAIL is a member of the TNF superfamily that has been firstly studied and evaluated for its anti-cancer activity, and the insights into its biology have already led to the identification of several TRAIL-based anticancer strategies with strong clinical therapeutic potentials. Nonetheless, the TRAIL system is far more complex and it can lead to a wider range of biological effects other than the ability of inducing apoptosis in cancer cells. By virtue of the different receptors and the different signalling pathways involved, TRAIL plays indeed a role in the regulation of different processes of the innate and adaptive immune system and this feature makes it an intriguing molecule under consideration in the development/progression/treatment of several immunological disorders. In this context, central nervous system represents a peculiar anatomic site where, despite its "status" of immune-privileged site, both innate and adaptive inflammatory responses occur and are involved in several pathological conditions. A number of studies have evaluated the role of TRAIL and of TRAIL-related pathways as pro-inflammatory or protective stimuli, depending on the specific pathological condition, confirming a twofold nature of this molecule. In this light, the aim of this review is to summarize the main preclinical evidences of the potential/involvement of TRAIL molecule and TRAIL pathways for the treatment of central nervous system disorders and the key suggestions coming from their assessment in preclinical models as proof of concept for future clinical studies.

Serum From Advanced Heart Failure Patients Promotes Angiogenic Sprouting and Affects the Notch Pathway in Human Endothelial Cells.

It is unknown whether components present in heart failure (HF) patients' serum provide an angiogenic stimulus. We sought to determine whether serum from HF patients affects angiogenesis and its major modulator, the Notch pathway, in human umbilical vein endothelial cells (HUVECs). In cells treated with serum from healthy subjects or from patients at different HF stage we determined: (1) Sprouting angiogenesis, by measuring cells network (closed tubes) in collagen gel. (2) Protein levels of Notch receptors 1, 2, 4, and ligands Jagged1, Delta-like4. We found a higher number of closed tubes in HUVECs treated with advanced HF patients serum in comparison with cells treated with serum from mild HF patients or controls. Furthermore, as indicated by the reduction of the active form of Notch4 (N4IC) and of Jagged1, advanced HF patients serum inhibited Notch signalling in HUVECs in comparison with mild HF patients' serum and controls. The circulating levels of NT-proBNP (N-terminal of the pro-hormone brain natriuretic peptide), a marker for the detection and evalutation of HF, were positively correlated with the number of closed tubes (r = 0.485) and negatively with Notch4IC and Jagged1 levels in sera-treated cells (r = -0.526 and r = -0.604, respectively). In conclusion, we found that sera from advanced HF patients promote sprouting angiogenesis and dysregulate Notch signaling in HUVECs. Our study provides in vitro evidence of an angiogenic stimulus arising during HF progression and suggests a role for the Notch pathway in it. J. Cell. Physiol. 231: 2700-2710, 2016. © 2016 Wiley Periodicals, Inc.

Selective induction of TP53I3/p53-inducible gene 3 (PIG3) in myeloid leukemic cells, but not in normal cells, by Nutlin-3.

The small molecule inhibitor of the MDM2/p53 interaction Nutlin-3 is a promising anti-cancer agent, which exhibits activity against a variety of cancers, including acute myeloid leukemia (AML). Previous studies have shown that Nutlin-3 variably induces apoptosis and cell cycle arrest in cancer cells while it shows low/absent cytotoxicity in normal cells. However, the reason for the selective pro-apoptotic activity in cancer cells with respect to normal counterparts is incompletely understood. In this study, we have compared the induction of several known target genes of p53 in two p53(wild-type) AML cell lines, OCI-AML3 and MOLM, in comparison with primary normal peripheral blood mononuclear cells (PBMC). Among several p53-target genes activated both in AML cell lines and normal PBMC (BBC3, BAX, MDM2, FAS, CDKN1A, GDF15, GADD45A, TNFRSF10B, TP53I3/PIG3), only TP53I3/PIG3 was selectively activated in MOLM and OCI-AML3, but not in PBMC. The important role of TP53I3/PIG3 in mediating the apoptotic activity of Nutlin-3 was underlined by knock-down experiments with siRNA specific for TP53I3/PIG3, which resulted in a significant decrease in the pro-apoptotic activity of Nutlin-3.

In vitro endothelial cell proliferation assay reveals distinct levels of proangiogenic cytokines characterizing sera of healthy subjects and of patients with heart failure.

Although myocardial angiogenesis is thought to play an important role in heart failure (HF), the involvement of circulating proinflammatory and proangiogenic cytokines in the pathogenesis and/or prognosis of HF has not been deeply investigated. By using a highly standardized proliferation assay with human endothelial cells, we first demonstrated that sera from older (mean age 52 ± 7.6 years; n = 46) healthy donors promoted endothelial cell proliferation to a significantly higher extent compared to sera obtained from younger healthy donors (mean age 29 ± 8.6 years; n = 20). The promotion of endothelial cell proliferation was accompanied by high serum levels of several proangiogenic cytokines. When we assessed endothelial cell proliferation in response to HF patients' sera, we observed that a subset of sera (n = 11) promoted cell proliferation to a significantly lesser extent compared to the majority of sera (n = 18). Also, in this case, the difference between the patient groups in the ability to induce endothelial cell proliferation correlated to significant (P < 0.05) differences in serum proangiogenic cytokine levels. Unexpectedly, HF patients associated to the highest endothelial proliferation index showed the worst prognosis as evaluated in terms of subsequent cardiovascular events in the follow-up, suggesting that high levels of circulating proangiogenic cytokines might be related to a worse prognosis.

Nutlin-3 Loaded Ethosomes and Transethosomes to Prevent UV-Associated Skin Damage.

The skin's protective mechanisms, in some cases, are not able to counteract the destructive effects induced by UV radiations, resulting in dermatological diseases, as well as skin aging. Nutlin-3, a potent drug with antiproliferative activity in keratinocytes, can block UV-induced apoptosis by activation of p53. In the present investigation, ethosomes and transethosomes were designed as delivery systems for nutlin-3, with the aim to protect the skin against UV damage. Vesicle size distribution was evaluated by photon correlation spectroscopy and morphology was investigated by cryogenic transmission electron microscopy, while nutlin-3 entrapment capacity was evaluated by ultrafiltration and HPLC. The in vitro diffusion kinetic of nutlin-3 from ethosomes and transethosomes was studied by Franz cell. Moreover, the efficiency of ethosomes and transethosomes in delivering nutlin-3 and its protective role were evaluated in ex vivo skin explants exposed to UV radiations. The results indicate that ethosomes and transethosomes efficaciously entrapped nutlin-3 (0.3% w/w). The ethosome vesicles were spherical and oligolamellar, with a 224 nm mean diameter, while in transethosome the presence of polysorbate 80 resulted in unilamellar vesicles with a 146 nm mean diameter. The fastest nutlin-3 kinetic was detected in the case of transethosomes, with permeability coefficients 7.4-fold higher, with respect to ethosomes and diffusion values 250-fold higher, with respect to the drug in solution. Ex vivo data suggest a better efficacy of transethosomes to promote nutlin-3 delivery within the skin, with respect to ethosomes. Indeed, nutlin-3 loaded transethosomes could prevent UV effect on cutaneous metalloproteinase activation and cell proliferative response.

Characterization Methods for Nanoparticle-Skin Interactions: An Overview.

Research progresses have led to the development of different kinds of nanoplatforms to deliver drugs through different biological membranes. Particularly, nanocarriers represent a precious means to treat skin pathologies, due to their capability to solubilize lipophilic and hydrophilic drugs, to control their release, and to promote their permeation through the stratum corneum barrier. A crucial point in the development of nano-delivery systems relies on their characterization, as well as in the assessment of their interaction with tissues, in order to predict their fate under in vivo administration. The size of nanoparticles, their shape, and the type of matrix can influence their biodistribution inside the skin strata and their cellular uptake. In this respect, an overview of some characterization methods employed to investigate nanoparticles intended for topical administration is presented here, namely dynamic light scattering, zeta potential, scanning and transmission electron microscopy, X-ray diffraction, atomic force microscopy, Fourier transform infrared and Raman spectroscopy. In addition, the main fluorescence methods employed to detect the in vitro nanoparticles interaction with skin cell lines, such as fluorescence-activated cell sorting or confocal imaging, are described, considering different examples of applications. Finally, recent studies on the techniques employed to determine the nanoparticle presence in the skin by ex vivo and in vivo models are reported.

SRP-35, a newly identified protein of the skeletal muscle sarcoplasmic reticulum, is a retinol dehydrogenase.

In the present study we provide evidence that SRP-35, a protein we identified in rabbit skeletal muscle sarcoplasmic reticulum, is an all-trans-retinol dehydrogenase. Analysis of the primary structure and tryptic digestion revealed that its N-terminus encompasses a short hydrophobic sequence bound to the sarcoplasmic reticulum membrane, whereas its C-terminal catalytic domain faces the myoplasm. SRP-35 is also expressed in liver and adipocytes, where it appears in the post-microsomal supernatant; however, in skeletal muscle, SRP-35 is enriched in the longitudinal sarcoplasmic reticulum. Sequence comparison predicts that SRP-35 is a short-chain dehydrogenase/reductase belonging to the DHRS7C [dehydrogenase/reductase (short-chain dehydrogenase/reductase family) member 7C] subfamily. Retinol is the substrate of SRP-35, since its transient overexpression leads to an increased production of all-trans-retinaldehyde. Transfection of C2C12 myotubes with a fusion protein encoding SRP-35-EYFP (enhanced yellow fluorescent protein) causes a decrease of the maximal Ca²âº released via RyR (ryanodine receptor) activation induced by KCl or 4-chloro-m-chresol. The latter result could be mimicked by the addition of retinoic acid to the C2C12 cell tissue culture medium, a treatment which caused a significant reduction of RyR1 expression. We propose that in skeletal muscle SRP-35 is involved in the generation of all-trans-retinaldehyde and may play an important role in the generation of intracellular signals linking Ca2+ release (i.e. muscle activity) to metabolism.

Nutrients, herbal bioactive derivatives and commensal microbiota as tools to lower the risk of SARS-CoV-2 infection.

The SARS-CoV-2 outbreak has infected a vast population across the world, causing more than 664 million cases and 6.7 million deaths by January 2023. Vaccination has been effective in reducing the most critical aftermath of this infection, but some issues are still present regarding re-infection prevention, effectiveness against variants, vaccine hesitancy and worldwide accessibility. Moreover, although several old and new antiviral drugs have been tested, we still lack robust and specific treatment modalities. It appears of utmost importance, facing this continuously growing pandemic, to focus on alternative practices grounded on firm scientific bases. In this article, we aim to outline a rigorous scientific background and propose complementary nutritional tools useful toward containment, and ultimately control, of SARS-CoV-2 infection. In particular, we review the mechanisms of viral entry and discuss the role of polyunsaturated fatty acids derived from α-linolenic acid and other nutrients in preventing the interaction of SARS-CoV-2 with its entry gateways. In a similar way, we analyze in detail the role of herbal-derived pharmacological compounds and specific microbial strains or microbial-derived polypeptides in the prevention of SARS-CoV-2 entry. In addition, we highlight the role of probiotics, nutrients and herbal-derived compounds in stimulating the immunity response.

Activation of the p53 pathway induces α-smooth muscle actin expression in both myeloid leukemic cells and normal macrophages.

A range of cell types of mesenchymal origin express α-smooth muscle actin (α-SMA), a protein that plays a key role in controlling cell motility and differentiation along the fibrocyte and myofibroblast lineages. Although α-SMA is often expressed in stromal cells associated to a variety of cancers including hematological malignancies, up to now the role of anti-cancer drugs on α-SMA has not been deeply investigated. In this study, we demonstrated that Nutlin-3, the small molecule inhibitor of the MDM2/p53 interactions, significantly up-regulated the mRNA and protein levels of α-SMA in normal macrophages as well as in p53(wild-type) but not in p53(mutated/null) myeloid leukemic cells. The p53-dependence of α-SMA up-regulation induced by Nutlin-3 was demonstrated in experiments performed with siRNA for p53. Of note, Nutlin-3 mediated up-regulation of α-SMA in OCI leukemic cells was accompanied by cell adhesion to plastic substrate and by reduced cell migratory response in transwell assays. Notably, the role of α-SMA induction in the modulation of myeloid cell migration was clearly documented in α-SMA gene knockdown experiments. In addition, Nutlin-3 significantly up-regulated α-SMA expression in primary endothelial cells, but not in fibroblasts and mesenchymal stem cells (MSC). Conversely, transforming growth factor-ß1 up-regulated α-SMA in fibroblasts and MSC, but not in macrophages and endothelial cells. Taken together, these data indicate that Nutlin-3 is a potent inducer of α-SMA in both normal and leukemic myeloid cells as well as in endothelial cells.

Anti-leukemic activity of dasatinib in both p53(wild-type) and p53(mutated) B malignant cells.

The multi-kinase inhibitor dasatinib induced a variable but significant decrease of viability in both p53(wild-type) (EHEB, JVM-2, JVM-3) and p53(mutated) (MEC-1, MEC-2, BJAB) prolymphocytic B leukemic cells, due to a combination of cell cycle block in G1 and apoptosis. Antibody phospho-kinase array analysis revealed that dasatinib inhibited the phosphorylation of various kinases, including ERK1/2 and p38/MAPK as well as of STAT3 transcription factors, in both p53(wild-type) and p53(mutated) cells. Therefore, dasatinib might offer a novel therapeutic strategy not only for p53(wild-type), but also for p53(mutated) B malignancies that have the worst prognosis and urgently need innovative therapeutic approaches.

The sorafenib plus nutlin-3 combination promotes synergistic cytotoxicity in acute myeloid leukemic cells irrespectively of FLT3 and p53 status.

BACKGROUND: Both the multi-kinase inhibitor sorafenib and the small molecule inhibitor of the MDM2/p53 interaction, nutlin-3, used alone, have shown promising anti-leukemic activity in acute myeloid leukemia cells. Thus, in this study we investigated the effect of the combination of sorafenib plus nutlin-3 in acute myeloid leukemia. DESIGN AND METHODS: Primary acute myeloid leukemia blasts (n=13) and FLT3(wild-type)/p53(wild-type) (OCI-AML3), FLT3(mutated)/p53(wild-type) (MOLM), FLT3(mutated)/p53(mutated) (MV4-11), FLT3(wild-type)/p53(deleted) (HL60) or FLT3(wild-type)/p53(mutated) (NB4) acute myeloid cell lines were exposed to sorafenib, used alone or in association with nutlin-3 at a 1:1 ratio, in a range of clinically achievable concentrations (1-10 µM). Induction of apoptosis and autophagy was evaluated by transmission electron microscopy and by specific flow cytometry analyses. The levels of Mcl-1, p53 and Bak proteins were analyzed by western blotting. Knock-down of Bax and Bak gene expression was performed in transfection experiments with specific short interfering RNA. RESULTS: The sorafenib+nutlin-3 drug combination exhibits synergistic cytotoxicity in primary acute myeloid leukemia blasts and in acute myeloid leukemia cell lines with maximal cytotoxicity in FLT3(mutated) MV4-11 and MOLM, followed by the FLT3(wild-type) OCI-AML3, HL60 and NB4 cell lines. The cytotoxic activity of sorafenib+nutlin-3 was characterized by an increase of both apoptosis and autophagy. Moreover, Bax and Bak showed prominent roles in mediating the decrease of cell viability in response to the drug combination in p53(wild-type) OCI-AML3 and p53(deleted) HL-60 cells, respectively, as demonstrated in transfection experiments performed with specific short interfering RNA. CONCLUSIONS: Our data demonstrate that acute myeloid leukemia cells show a variable but overall good susceptibility to the innovative therapeutic combination of sorafenib+nutlin-3, which differentially involves the pro-apoptotic Bcl-2 family members Bax and Bak in p53(wild-type) and p53(deleted) cells.

Therapeutic potential of the MDM2 inhibitor Nutlin-3 in counteracting SARS-CoV-2 infection of the eye through p53 activation.

Starting from the beginning of the severe acute respiratory syndrome Coronavirus-2 (SARS-CoV-2) global pandemic, most of the published data has concentrated on the respiratory signs and symptoms of Covid-19 infection, underestimating the presence and importance of ocular manifestations, such as conjunctivitis, usually reported in SARS-CoV-2 infected patients. With the present review we intend to resume the ocular involvement in SARS-CoV-2 infection and the recent discoveries about the different cell types and tissues of the eye that can be directly infected by SARS-CoV-2 and propagate the infection. Moreover, reviewing literature data about p53 expression in normal and diseased eye tissues, we hypothesize that the pleiotropic protein p53 present at high levels in cornea, conjunctiva and tear film might play a protective role against SARS-CoV-2 infection. Since p53 can be easily up-regulated by using small molecule non-genotoxic inhibitors of MDM2, we propose that topical use of Nutlin-3, the prototype member of MDM2 inhibitors, might protect the anterior surface of the eye from SARS-CoV-2 infection, reducing the spreading of the virus.

MDM2 inhibitors-mediated disruption of mitochondrial metabolism: A novel therapeutic strategy for retinoblastoma.

MDM2 is the principal inhibitor of p53, and MDM2 inhibitors can disrupt the physical interaction between MDM2 and p53. The half-life of p53 is very short in normal cells and tissues, and an uncontrolled increase in p53 levels has potential harmful effects. It has been shown that p53 is frequently mutated in most cancers; however, p53 mutations are rare in retinoblastoma. Therefore, therapeutic strategies aimed at increasing the expression levels of wild-type p53 are attractive. In this minireview, we discuss the potential use of nutlin-3, the prototype small molecule inhibitor that disrupts the MDM2-p53 interaction, for the treatment of retinoblastoma. Although p53 has pleiotropic biological effects, the functions of p53 depend on its sub-cellular localization. In the nucleus, p53 induces the transcription of a vast array of genes, while in mitochondria, p53 regulates mitochondrial metabolism. This review also discusses the relative contribution of p53-mediated gene transcription and mitochondrial perturbation for retinoblastoma treatment.

Cell cycle block by p53 activation reduces SARS-CoV-2 release in infected alveolar basal epithelial A549-hACE2 cells.

SARS-CoV viruses have been shown to downregulate cellular events that control antiviral defenses. They adopt several strategies to silence p53, key molecule for cell homeostasis and immune control, indicating that p53 has a central role in controlling their proliferation in the host. Specific actions are the stabilization of its inhibitor, MDM2, and the interference with its transcriptional activity. The aim of our work was to evaluate a new approach against SARS-CoV-2 by using MDM2 inhibitors to raise p53 levels and activate p53-dependent pathways, therefore leading to cell cycle inhibition. Experimental setting was performed in the alveolar basal epithelial cell line A549-hACE2, expressing high level of ACE2 receptor, to allow virus entry, as well as p53 wild-type. Cells were treated with several concentrations of Nutlin-3 or RG-7112, two known MDM2 inhibitors, for the instauration of a cell cycle block steady-state condition before and during SARS-CoV-2 infection, and for the evaluation of p53 activation and impact on virus release and related innate immune events. The results indicated an efficient cell cycle block with inhibition of the virion release and a significant inhibition of IL-6, NF-kB and IFN-λ expression. These data suggest that p53 is an efficient target for new therapies against the virus and that MDM2 inhibitors deserve to be further investigated in this field.

Perifosine selectively induces cell cycle block and modulates retinoblastoma and E2F1 protein levels in p53 mutated leukemic cell lines.

The effect of the single-chain alkylphospholipid perifosine was analyzed in p53(wild-type) (SKW6.4, OCI and MOLM), p53(mutated) (BJAB, MAVER) and p53(null) (HL-60) leukemic cell lines. Perifosine promoted cytotoxicity with a combination of apoptosis induction in all cell lines and cell cycle block at the G(2)M checkpoint, which was selectively observed in p53(mutated) BJAB and MAVER cell lines. At the molecular level, perifosine induced hypophosphorylation of retinoblastoma protein and the degradation of E2F1 protein in p53(mutated) but not in p53(wild-type) cells. These data indicate that perifosine potentially represents an innovative therapeutic approach for p53(mutated) hematological malignancies.

Overcoming of Microenvironment Protection on Primary Chronic Lymphocytic Leukemia Cells after Treatment with BTK and MDM2 Pharmacological Inhibitors.

In B-chronic lymphocytic leukemia (B-CLL), the interaction between leukemic cells and the microenvironment promotes tumor cell survival. The Bruton's tyrosine kinase (BTK) inhibitor ibrutinib is one of the first-in-class molecules for the treatment of B-CLL patients; however, the emerging mechanisms of resistance to ibrutinib call for new therapeutic strategies. The purpose of the current study was to investigate the ability of ibrutinib plus the MDM2-inhibitor nutlin-3 to counteract the tumor microenvironment protective effect. We observed that primary B-CLL cells cultivated in microenvironment mimicking conditions were protected from apoptosis by the up-regulation of c-MYC and of p53. In the same setting, combined treatments with ibrutinib plus nutlin-3 led to significantly higher levels of apoptosis compared to the single treatments, counteracting the c-MYC up-regulation. Moreover, the combination induced high p53 levels and a significant dissipation of the mitochondrial membrane potential, together with BAX cleavage in the more active p18 form and phospho-BAD down-regulation, that are key components of the mitochondrial apoptotic pathway, enhancing the apoptosis level. Our findings propose a new therapeutic strategy to overcome the tumor microenvironment protection involved in B-CLL resistance to drugs, with possible clinical implications also for other hematologic and solid tumors for which ibrutinib is considered a therapeutic option.

Human full-length osteoprotegerin induces the proliferation of rodent vascular smooth muscle cells both in vitro and in vivo.

BACKGROUND/AIMS: Since elevated plasma levels of osteoprotegerin (OPG) represent a risk factor for death and heart failure in patients affected by diabetes mellitus and coronary artery disease, this study aimed to elucidate potential roles of OPG in the pathogenesis of atherosclerosis. METHODS AND RESULTS: Recombinant human full-length OPG, used at concentrations comparable to the elevated levels found in the serum of diabetic patients, significantly increased the proliferation rate of rodent vascular smooth muscle cells (VSMC). To mimic the moderate chronic elevation of OPG observed in diabetic patients, low doses (1 microg/mouse) of full-length human OPG were injected intraperitoneally every 3 weeks in diabetic apolipoprotein E (apoE)-null mice. The group of animals treated for 12 weeks with recombinant OPG showed a small increase in the total aortic plaque area at necropsy in comparison to vehicle-treated animals. Importantly, while no differences in the amount of interstitial collagen or the degree of macrophage infiltration were observed between OPG-treated and vehicle-treated apoE-null diabetic animals, a significant increase in the number of alpha-actin-positive smooth muscle cells was observed in the plaques of OPG-treated mice. CONCLUSIONS: Our data suggest that OPG promotes VSMC proliferation and might be directly involved in pathogenetic aspects of atherosclerosis.