Maria Castellano

In vivo activity of miR-34a mimics delivered by stable nucleic acid lipid particles (SNALPs) against multiple myeloma.

Multiple myeloma (MM) is a disease with an adverse outcome and new therapeutic strategies are urgently awaited. A rising body of evidence supports the notion that microRNAs (miRNAs), master regulators of eukaryotic gene expression, may exert anti-MM activity. Here, we evaluated the activity of synthetic miR-34a in MM cells. We found that transfection of miR-34a mimics in MM cells induces a significant change of gene expression with relevant effects on multiple signal transduction pathways. We detected early inactivation of pro-survival and proliferative kinases Erk-2 and Akt followed at later time points by caspase-6 and -3 activation and apoptosis induction. To improve the in vivo delivery, we encapsulated miR-34a mimics in stable nucleic acid lipid particles (SNALPs). We found that SNALPs miR-34a were highly efficient in vitro in inhibiting growth of MM cells. Then, we investigated the activity of the SNALPs miR-34a against MM xenografts in SCID mice. We observed significant tumor growth inhibition (p<0.05) which translated in mice survival benefits (p = 0.0047). Analysis of miR-34a and NOTCH1 expression in tumor retrieved from animal demonstrated efficient delivery and gene modulation induced by SNALPs miR-34a in the absence of systemic toxicity. We here therefore provide evidence that SNALPs miR-34a may represent a promising tool for miRNA-therapeutics in MM.

Evaluation of the in vitro and in vivo antiangiogenic effects of denosumab and zoledronic acid

Denosumab (Dmab) and zoledronic acid (ZOL) are antiresorptive agents, with different mechanisms of action, that are indicated for delaying the onset of skeletal-related events in patients with bone metastases from solid tumors. Clinical and preclinical data suggest that ZOL may have also anti-angiogenic activity; however, the effects of Dmab (a fully humanized antibody against the receptor activator of nuclear factor kappa B ligand) on angiogenesis are largely unknown. The objective of this study was to compare the potential anti-angiogenic activity of Dmab with that of ZOL in preclinical models. Dmab (0.31 to 160 μM) had no effect on the viability of human MDA-MB-436 and CG5 breast cancer cells or human umbilical vein endothelial cells (HUVECs) and no effect on tubule formation or invasion of HUVECs. In contrast, ZOL (0.31 to 160 μM) decreased the viability of breast cancer and HUVECs in a time- and concentration-dependent manner and also inhibited HUVEC tubule formation and invasion. In vivo, ZOL (20 μg/mouse for three times a week for three consecutive weeks) inhibited angiogenesis in Matrigel plugs and inhibited the growth and neo-angiogenesis of CG5 xenografts in athymic nude mice. In contrast, Dmab (10 mg/Kg twice a week for 4 consecutive weeks) had no effect on Matrigel vascularization or xenograft growth in this model. These findings support the potential antiangiogenic and anticancer activity of ZOL in vitro and in vivo and further suggest that Dmab does not have antiangiogenic activity. Additional studies are needed to elucidate the potential anticancer activity of Dmab.

Structural and Functional Alterations of Cellular Components as Revealed by Electron Microscopy

Scanning (SEM) and transmission electron microscopy (TEM) are two fundamental microscopic techniques widely applied in biological research for the study of ultrastructural cell components. With these methods, especially TEM, it is possible to detect and quantify the morphological and ultrastructural parameters of intracellular organelles (mitochondria, Golgi apparatus, lysosomes, peroxisomes, endosomes, endoplasmic reticulum, cytoskeleton, nucleus, etc.) in normal and pathological conditions. The study of intracellular vesicle compartmentalization is raising even more interest in the light of the importance of intracellular localization of mediators of the signaling in eliciting different biological responses. The study of the morphology of some intracellular organelles can supply information on the bio‐energetic status of the cells. TEM has also a pivotal role in the determination of different types of programmed cell death. In fact, the visualization of autophagosomes and autophagolysosomes is essential to determine the occurrence of autophagy (and also to discriminate micro‐autophagy from macro‐autophagy), while the presence of fragmented nuclei and surface blebbing is characteristic of apoptosis. SEM is particularly useful for the study of the morphological features of the cells and, therefore, can shed light, for instance, on cell–cell interactions. After a brief introduction on the basic principles of the main electron microscopy methods, the article describes some cell components with the aim to demonstrate the huge role of the ultrastructural analysis played in the knowledge of the relationship between function and structure of the biological objects. Microsc. Res. Tech., 76:1057–1069, 2013.

Cyclohexa-2,5-diene-1,4-dione-based antiproliferative agents: design, synthesis, and cytotoxic evaluation

BackgroundTumors are diseases characterized by uncontrolled cell growth and, in spite of the progress of medicine over the years, continue to represent a major threat to the health, requiring new therapies. Several synthetic compounds, such as those derived from natural sources, have been identified as anticancer drugs; among these compounds quinone represent the second largest class of anticancer agents in use. Several studies have shown that these act on tumor cells through several mechanisms. An important objective of this work is to develop quinoidscompounds showing antitumor activity, but with fewer side effects. The parachinone cannabinol HU-331, is a small molecule that with its core 4-hydroxy-1,4-benzoquinone, exhibits a potent and selective cytotoxic activity on different tumor cell lines. A series of derivatives 3-hydroxy-1,4-benzochinoni were thus developed through HU-331 chemical modifications. The purpose of the work is to test the ability of the compounds to induce proliferative inhibition and study the mechanisms of cell death.MethodsThe antitumor activities were evaluated in vitro by examining their cytotoxic effects against different human cancer cell lines. All cell lines tested were plated in 96-multiwell and treated with HU-100-V at different concentrations and cell viability was evaluated byMTT assay. Subsequently via flow cytometry (FACS) it was possible to assess apoptosis by the system of double labeling with PI and Annexin-V, and the effect of the compounds on ROS formation by measuring the dichlorofluorescein fluorescence.ResultsThe substitution by n-hexyl chain considerably enhanced the bioactivity of the compounds. In details, 2-hexyl-5-hydroxycyclohexa-2,5-diene-1,4-dione (V), 2,5-Dimethoxy-3-hexyl-2,5-cyclohexadiene-1,4-dione (XII) and 2-hydroxy-5-methoxy-3-hexyl-cyclohexa-2,5-diene-1,4-dione (XIII) showed most prominent cytotoxicity against almost human tumour cell lines. Compound V was further subjected to downstream apoptotic analysis, demostrating a time-dependent pro-apoptotic activity on human melanoma M14 cell line mediated by caspases activation and poly-(ADP-ribose)-polymerase (PARP) protein cleavage.ConclusionsThese findings indicate that 2-hexyl-5-idrossicicloesa-2,5-diene-1,4-dione can be a promising compound for the design of a new class of antineoplastic derivatives.Carmen Petronzi, Michela Festa, Antonella Peduto and Maria Castellano: equally contributed equally to this work.

INTERLEUKIN-2 AND LANREOTIDE IN THE TREATMENT OF MEDULLARY THYROID CANCER: IN VITRO AND IN VIVO STUDIES

CONTEXT To date no efficacious treatments are available for advanced medullary thyroid carcinoma (MTC). OBJECTIVE We investigated in vitro and in vivo a new strategy for the therapy of MTC, combining human recombinant IL-2 with lanreotide (LAN), a somatostatin analog. METHODS The in vitro effects of LAN on the sensitivity of TT cells, a MTC cell line, to IL-2-stimulated human peripheral blood mononuclear cells were determined by a lactate dehydrogenase release assay. In addition, we evaluated the toxicity, the effects on quality of life, and the antitumor activity of sc low-dose IL-2 in combination with LAN (90 mg every 28 days) in a series of 6 patients with symptomatic and advanced MTC. RESULTS The cytotoxicity of IL-2-activated peripheral blood mononuclear cells was significantly increased in TT cells treated with LAN or LAN plus IL-2 compared with that in TT cells without treatment. The therapy was well tolerated, and a statistically significant improvement of quality of life was observed in patients treated with the combination of LAN and IL-2. After 6 months of therapy, partial response and stable disease have been recorded in 2 and 3 patients, respectively, with a significant decrease in calcitonin levels in 3 patients. CONCLUSIONS Both in vitro and in vivo evidence suggests that the combination of LAN and IL-2 may have a role in the management of advanced and symptomatic MTC. However, these preliminary data require further validation in larger randomized trials.

Identification of a research protocol to study orthodontic tooth movement

Aim: The orthodontic movement is associated with a process of tissue remodeling together with the release of several chemicalmediators in periodontal tissues. Each mediator is a potential marker of tooth movement and expresses biological processes as: tissueinflammation and bone remodeling. Different amounts of every mediator are present in several tissues and fluids of the oral cavity.Therefore, there are different methods that allow sampling with several degrees of invasiveness. Chemical mediators are alsosubstances of different molecular nature, and multiple kind of analysis methods allow detection. The purpose of this study was to draftthe best research protocol for an optimal study on orthodontic movement efficiency.Methods: An analysis of the international literature have been made, to identify the gold standard of each aspect of the protocol: typeof mediator, source and method of sampling and analysis method.Results: From the analysis of the international literature was created an original research protocol for the study and the assessment ofthe orthodontic movement, by using the biomarkers of the tooth movement.Conclusions: The protocol created is based on the choice of the gold standard of every aspect already analyzed in the literature and inexisting protocols for the monitoring of orthodontic tooth movement through the markers of tooth movement. Clinical trials rerequired for the evaluation and validation of the protocol created.