Nanoparticles loaded with Nutlin-3 display cytotoxicity towards p53(wild-type) JVM-2 but not towards p53(mutated) BJAB leukemic cells.

The small molecule Nutlin-3 is a potent antagonist of the murine double minute 2 (MDM2)/p53 interaction exhibiting promising therapeutic anti-cancer activity. Nutlin-3 has been proposed as an anti-neoplastic agent for the treatment of onco-hematological diseases characterized by a lower incidence of p53 mutation with respect to solid tumors. Indeed, based on its selective non-genotoxic p53 activation, Nutlin-3 might represent an alternative to the current cytotoxic chemotherapy. To overcome the poor bioavailability of Nutlin-3, we have assessed the potential efficacy of Nutlin-3 loaded poly(lactide-co-glycolide) (PLGA) nanoparticles (NP) against hematological malignancies. To test the specificity of the anti-leukemic activity, we have used leukemic cell lines characterized by different p53 status (JVM-2 and BJAB). NP loaded with Nutlin-3 (NP-Nutlin) were rapidly taken up by the leukemic cells and were as effective as native Nutlin-3 in promoting both induction of apoptosis and cell cycle arrest in p53(wild-type) JVM-2 cells, but not in p53(mutated) BJAB cells. Moreover, injection of NP-Nutlin, but not of free Nutlin-3, in a JVM-2-derived xenograft mouse model, reduced the subcutaneous tumor volume and promoted induction of apoptosis in the tumor mass. Overall, the chemical and structural characteristics of the NP-Nutlin-3, as well as their biological activity in vitro and in vivo, made them promising for further preclinical evaluations as potentially useful anti-leukemic carriers.

MDM2 non-genotoxic inhibitors as innovative therapeutic approaches for the treatment of pediatric malignancies.

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

State of the art of the therapeutic perspective of sorafenib against hematological malignancies.

The bi-aryl urea multi-kinase inhibitor Sorafenib (BAY 43-9006, Nexavar) was initially approved for the treatment of unresectable hepatocellular carcinoma and advanced renal cell carcinoma. Eleven years after its first description in PubMed, the therapeutic potential of Sorafenib has been evaluated in an increasing number of studies, mainly focused on solid tumors. More recently, the potential usefullness of Sorafenib has started to emerge also against hematological malignancies. At the molecular level, besides the RAF kinase pathway, which represents the first therapeutic target of Sorafenib, additional kinases, in particular the vascular endothelial growth factor receptor, have been identified as important targets of Sorafenib. A great interest for the potential use of Sorafenib against acute myeloid leukemia (AML) arose when it was demonstrated that a specific mutation of a kinase gene, called FMS-like tyrosin-kinase-3- internal tandem duplication (FLT-3-ITD) and occurring in more than 30% of AML, represents a molecular target of Sorafenib. However, recent phase I and II clinical studies showed that, in spite of its ability to suppress the activity of this mutated kinase, resistence to Sorafenib rapidly occurs in AML, suggesting that Sorafenib will be more effective in combined therapy than used as single drug. Another critical molecular target of Sorafenib is the anti-apoptotic protein Mcl-1. The ability of Sorafenib to rapidly shut-off Mcl-1 in virtually all the hematological malignancies investigated, including the B-chronic lymphocytic leukemia, represents a key element for its antileukemic activity as well as for therapeutic combinations based on Sorafenib. In this respect, it is of particular interest that many chemotherapeutic drugs or innovative anti-neoplastic compounds, such as recombinant TRAIL or inibitors of MDM2 protein, are either unable to down-regulate Mcl-1 or in some instances promote a paradoxical induction of Mcl-1. In this review, the growing evidences for the role of Mcl-1 in mediating the anti-leukemic activity of Sorafenib will be discussed in relationship with promising therapeutic perspectives.

Activation of PKC-epsilon counteracts maturation and apoptosis of HL-60 myeloid leukemic cells in response to TNF family members.

Protein kinase C (PKC)-epsilon, a component of the serine/threonine PKC family, has been shown to influence the survival and differentiation pathways of normal hematopoietic cells. Here, we have modulated the activity of PKC-epsilon with specific small molecule activator or inhibitor peptides. PKC-epsilon inhibitor and activator peptides showed modest effects on HL-60 maturation when added alone, but PKC-epsilon activator peptide significantly counteracted the pro-maturative activity of tumor necrosis factor (TNF)-alpha towards the monocytic/macrophagic lineage, as evaluated in terms of CD14 surface expression and morphological analyses. Moreover, while PKC-epsilon inhibitor peptide showed a reproducible increase of TNF-related apoptosis inducing ligand (TRAIL)-induced apoptosis, PKC-epsilon activator peptide potently counteracted the pro-apoptotic activity of TRAIL. Taken together, the anti-maturative and anti-apoptotic activities of PKC-epsilon envision a potentially important proleukemic role of this PKC family member.

Perspectives of protein kinase C (PKC) inhibitors as anti-cancer agents.

Although the role of serine/threonine protein kinase C (PKC) in malignant transformation is known from decades, an anti-PKC based approach in cancer therapy was hampered for the difficulties in developing pharmacological compounds able to selectively inhibit specific PKC isoforms. In this review, the role of PKC-epsilon and PKC-delta in promoting and counteracting tumor progression in different types of cancer, respectively, will be discussed in relationship with promising therapeutic perspectives based either on small molecule inhibitors or on natural compounds. Among a myriad of molecules able to modulate PKC activity, we will focus on the role of the enzastaurin and briostatin-1, which already entered clinical trials for several human cancers.

Activation of PKC-ε counteracts maturation and apoptosis of HL-60 myeloid leukemic cells in response to TNF family members.

Protein kinase C (PKC)-ε, a component of the serine/threo-nine PKC family, has been shown to influence the survival and differentiation pathways of normal hematopoietic cells. Here, we have modulated the activity of PKC-ε with specific small molecule activator or inhibitor peptides. PKC-ε inhibitor and activator peptides showed modest effects on HL-60 maturation when added alone, but PKC-ε activator peptide significantly counteracted the pro-maturative activity of tumor necrosis factor (TNF)-α towards the monocytic/macrophagic lineage, as evaluated in terms of CD14 surface expression and morphological analyses. Moreover, while PKC-ε inhibitor peptide showed a reproducible increase of TNF-related apoptosis inducing ligand (TRAIL)-induced apoptosis, PKC-ε activator peptide potently counteracted the pro-apoptotic activity of TRAIL. Taken together, the anti-maturative and anti-apoptotic activities of PKC-ε envision a potentially important proleukemic role of this PKC family member.

Characterization of HIV-1 Tat proteins mutated in the transactivation domain for prophylactic and therapeutic application.

Previous work from our group showed that genetic immunization of mice with HIV-1 tat genes (tat22 and tat22/37), encoding Tat proteins mutated in the transactivation domain and lacking Tat-transactivating activity, evoke an immune response to wild-type Tat, both humoral and cellular. In the present work we report that the mutated Tat proteins localize within the cells, are released and taken up by the cells in a fashion similar to wild-type Tat. Moreover, the exogenous mutated Tat proteins interfere with the transactivating function of extracellular wild-type Tat. These results support the notion that tat22 and tat22/37 genes may represent good candidates for the development of an anti-HIV-1 vaccine, especially for HIV-1 infected patients.

Tissue dose, DNA adducts, oxidative DNA damage and CYP1A-immunopositive proteins in mussels exposed to waterborne benzo[a]pyrene.

A collaborative study was performed on Mediterranean mussels (Mytilus galloprovincialis) exposed to a wide dose-range (0.5-1000 ppb) of benzo[a]pyrene (B[a]P). We selected this model polycyclic aromatic hydrocarbon in order to confirm the formation of a specific DNA adduct, previously detected in gill DNA, and to clarify the in vivo effects of this mutagenic chemical requiring host-metabolism in mussels. B[a]P concentration reached consistently higher values in the digestive gland than in other analyzed tissues of mussels exposed to B[a]P for 2 or 3 days. With the exception of some values at 1000 ppb of B[a]P. DNA adduct levels increased significantly with the dose in gills and digestive gland and ranged from 0.054 to 0.789 adducts per 10(8) nucleotides (mean values per dose-point). Conversely, more complex dose-response relationships were found by detecting in parallel the levels of an oxidative DNA lesion (8-OHdG) and of CYP1A-immunopositive proteins (the latter measured in the digestive gland only). Overall, the formation of DNA adducts, the evidence of oxidative DNA damage, and changes in CYP1A-immunopositive protein levels support the hypothesis that B[a]P can induce DNA damage in mussels through a number of different molecular mechanisms.