Serum Free Light-Chain Ratio at Diagnosis Is Associated with Early Renal Damage in Multiple Myeloma: A Case Series Real-World Study.

The serum free light-chain (FLC) ratio is a sensitive tool for the differential diagnosis of plasma cell disorders and is biomarker of multiple myeloma (MM) progression from premalignant conditions. Here, we investigate the potential role of FLC ratio at diagnosis in identifying early renal damage in MM patients and other correlations with clinical, laboratory, and molecular findings. A total of 34 MM patients who had undergone autologous stem cell transplantation were included in this retrospective case series study, and FLC quantification was performed with nephelometric assays. In our study, sFLC ratio was significantly associated with light-chain MM and ß-2 microglobulin levels, likely indicating a high disease burden at diagnosis, especially in patients without heavy chain M-protein at serum electrophoresis. Moreover, the sFLC ratio was inversely correlated with glomerular filtration rate, possibly identifying early renal damage in MM patients. Our preliminary results confirm the importance of early sFLC evaluation, especially in patients with the light-chain MM type and low disease burden, to minimize the risk of late renal failure.

Nanoparticle-based strategy for personalized B-cell lymphoma therapy.

B-cell lymphoma is associated with incomplete response to treatment, and the development of effective strategies targeting this disease remains challenging. A new personalized B-cell lymphoma therapy, based on a site-specific receptor-mediated drug delivery system, was developed in this study. Specifically, natural silica-based nanoparticles (diatomite) were modified to actively target the antiapoptotic factor B-cell lymphoma/leukemia 2 (Bcl2) with small interfering RNA (siRNA). An idiotype-specific peptide (Id-peptide) specifically recognized by the hypervariable region of surface immunoglobulin B-cell receptor was exploited as a homing device to ensure specific targeting of lymphoma cells. Specific nanoparticle uptake, driven by the Id-peptide, was evaluated by flow cytometry and confocal microscopy and was increased by approximately threefold in target cells compared with nonspecific myeloma cells and when a random control peptide was used instead of Id-peptide. The specific internalization efficiency was increased by fourfold when siRNA was also added to the modified nanoparticles. The modified diatomite particles were not cytotoxic and their effectiveness in downregulation of gene expression was explored using siRNA targeting Bcl2 and evaluated by quantitative real-time polymerase chain reaction and Western blot analyses. The resulting gene silencing observed is of significant biological importance and opens new possibilities for the personalized treatment of lymphomas.

New insights on the interaction between the isoforms 1 and 2 of human translation elongation factor 1A.

The eukaryotic translation elongation factor 1A (eEF1A) is a moonlighting protein that besides to its canonical role in protein synthesis is also involved in many other cellular processes such as cell survival and apoptosis. In a previous work, we identified eEF1A Raf-mediated phosphorylation sites and defined their role in the regulation of eEF1A half-life and apoptosis of human cancer cells. We proposed that the phosphorylation of eEF1A by C-Raf required the presence of both eEF1A isoforms thus suggesting the formation of a potential eEF1A heterodimer owning regulatory properties. This study aimed at investigating the cellular localization and interaction between two eEF1A isoforms. To this end, we developed chimera proteins by adding at the N-terminal end of both eEF1A1 and eEF1A2 cyan fluorescence protein (mCerulean) and yellow fluorescence protein (mVenus), respectively. The fluorescent eEF1A1 and eEF1A2 chimeras were both addressed to COS-7 cells and found co-localized in the cytoplasm at the level of cellular membranes. We highlighted FRET between the labeled N-termini of eEF1A isoforms. The intra-molecular FRET of this chimera was about 17%. Our results provide novel information on the intracellular distribution and interaction of eEF1A isoforms.

B-cell receptor-guided delivery of peptide-siRNA complex for B-cell lymphoma therapy.

BACKGROUND: Despite the clinical response of conventional anticancer therapy, including chemotherapeutic treatments, radiation therapy and corticosteroids, tumorigenic B-cell lymphomas show an incomplete response to clinical practices that result in a minimal residual disease (MRD) where few residual neoplastic cells undetected in vivo, replenish the cancer cell reservoir. This scenario, which is also shared with other cancer diseases, requires the development of strategies to advance in novel, selective targeting toward the tumorigenic cells that survive to the anticancer agents. METHODS: Here, we have taken advantage of the therapeutic properties of an idiotype specific peptide (pA20-36) that bind specifically to murine B-lymphoma cells in the setting of an anti cancer strategy, based on the selected delivery of electrostatic-based complex, peptide-siRNA. To this end, two engineered, arginine rich, peptides that included the pA20-36 targeting sequence were designed to bind fluorescent-labelled siRNA. One peptide presented 9 Arg at the C-terminal of pA20-36 whereas the other included 5 Arg at the N- and C-terminus, respectively. RESULTS: Compared to the control and random peptide-siRNA complexes, both pA20-36-siRNA complexes were endowed with the selective delivering of fluorescent-labelled siRNA toward the A20 murine B-cell lymphoma, as evaluated by cytofluorimetry and confocal microscopy, whereas fluorescent-labelled siRNA alone was not internalized in the selected cells. Compared to peptide controls, the use of the modified pA20-36 peptides complexed with siRNA anti-GAPDH and anti-Bcl2 showed a down-regulation in the expression levels of the corresponding genes. CONCLUSIONS: Peptide-siRNA complex can be suitable tool for both selective peptide-driven cell targeting and gene silencing. In this setting, the improvement of this strategy is expected to provide a safe and non-invasive approach for the delivery of therapeutic molecules.

A new strategy for label-free detection of lymphoma cancer cells.

In this paper, a new strategy for highly selective and sensitive direct detection of lymphoma cells by exploiting the interaction between a peptide and its B-cell receptor, has been evaluated. In particular, an idiotype peptide, able to specifically bind the B-cell receptor of A20 cells in mice engrafted with A20 lymphoma, has been used as molecular probe. The new detection technique has been demonstrated on a planar crystalline silicon chip. Coverage of 85% of silicon surface and detection efficiency of 8.5 × 10(-3) cells/µm(2) were obtained. The recognition strategy promises to extend its application in studying the interaction between ligands and their cell-surface receptors.

Diatomite biosilica nanocarriers for siRNA transport inside cancer cells.

BACKGROUND: Diatomite is a natural porous biomaterial of sedimentary origin, formed by fragments of diatom siliceous skeletons, called "frustules". Due to large availability in many areas of the world, chemical stability, and non-toxicity, these fossil structures have been widespread used in lot of industrial applications, such as food production, water extracting agent, production of cosmetics and pharmaceutics. However, diatomite is surprisingly still rarely used in biomedical applications. In this work, we exploit diatomite nanoparticles for small interfering ribonucleic acid (siRNA) transport inside human epidermoid cancer cells (H1355). METHODS: Morphology and composition of diatomite microfrustules (average size lower than 40µm) are investigated by scanning electron microscopy equipped by energy dispersive X-ray spectroscopy, Fourier transform infrared analysis, and photoluminescence measurements. Nanometric porous particles (average size lower than 450nm) are obtained by mechanical crushing, sonication, and filtering of micrometric frustules. siRNA bioconjugation is performed on both micrometric and nanometric fragments by silanization. RESULTS: In-vitro experiments show very low toxicity on exposure of the cells to diatomite nanoparticle concentration up to 300µg/ml for 72h. Confocal microscopy imaging performed on cancer cells incubated with siRNA conjugated nanoparticles demonstrates a cytoplasmatic localization of vectors. Gene silencing by delivered siRNA is also demonstrated. CONCLUSION: Our studies endorse diatomite nanoparticles as non-toxic nanocarriers for siRNA transport in cancer cells. GENERAL SIGNIFICANCE: siRNA is a powerful molecular tool for cancer treatment but its delivery is inefficient due to the difficulty to penetrate the cell membrane. siRNA-diatomite nanoconjugate may be well suited for delivery of therapeutic to cancer cells.

Diatomite silica nanoparticles for drug delivery.

Diatomite is a natural fossil material of sedimentary origin, constituted by fragments of diatom siliceous skeletons. In this preliminary work, the properties of diatomite nanoparticles as potential system for the delivery of drugs in cancer cells were exploited. A purification procedure, based on thermal treatments in strong acid solutions, was used to remove inorganic and organic impurities from diatomite and to make them a safe material for medical applications. The micrometric diatomite powder was reduced in nanoparticles by mechanical crushing, sonication, and filtering. Morphological analysis performed by dynamic light scattering and transmission electron microscopy reveals a particles size included between 100 and 300 nm. Diatomite nanoparticles were functionalized by 3-aminopropyltriethoxysilane and labeled by tetramethylrhodamine isothiocyanate. Different concentrations of chemically modified nanoparticles were incubated with cancer cells and confocal microscopy was performed. Imaging analysis showed an efficient cellular uptake and homogeneous distribution of nanoparticles in cytoplasm and nucleus, thus suggesting their potentiality as nanocarriers for drug delivery. PACS: 87.85.J81.05.Rm; 61.46. + w.

Raf kinases in signal transduction and interaction with translation machinery.

In recent years, a large amount of evidence has given a central role to translational control in diseases such as cancer, tissue hypertrophy and neurodegeneration. Its deregulation can directly modulate cell cycling, transformation and survival response. The aim of this review is to describe the interaction between Raf activation and the main characters of the translational machinery, such as the elongation factor 1A (eEF1A), which has been recognized in recent years as one of the most interesting putative oncogenes. A particular emphasis is given to an intriguing non-canonical role that eEF1A can play in the relationship between the Ras→Raf-1→MEK1→ERK-1/2 and PI3K→Akt signaling pathways. Recently, our group has described a C-Raf kinase-mediated phosphorylation of eEF1A triggered by a survival pathway induced upon interferon alpha (IFNα) treatment in the human epidermoid cancer cell line (H1355). This phosphorylation seems to be the center of the survival pathway that counteracts the well-known pro-apoptotic function of IFNα. Furthermore, we have identified two new phosphorylation sites on eEF1A (Ser21 and Thr88) that are substrates for Raf kinases in vitro and, likely, in vivo as well. These residues seem to have a significant functional role in the control of cellular processes, such as cell proliferation and survival. In fact, overexpression of eEF1A2 in gemcitabine-treated cancer cells caused the upregulation of phosphoAkt and an increase in cell viability, thereby suggesting that eEF1A2 could exert its oncogenic behavior by participating in the regulation of PI3K pathway.