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Properties of RNase A monomer and dimers depend on the oligomerization pathway followed.
No full textRNase A oligomerizatio
Influence of onconase in the therapeutic potential of PARP inhibitors in A375 malignant melanoma cells
No full textHuman malignant melanoma is one of the most aggressive cancers, accompanied with poor prognosis, metastatic evolution and high mortality. Many strategies have been developed using BRAF and MEK inhibitors in spite of the classic therapy with alkylating agents, but failure related to the ability of the tumor to activate alternative proliferation pathways occurred after promising initial successes. Poly(ADP-ribose) polymerase (PARP) enzymes are well known to be crucial for DNA damage response, and PARP inhibition results in the accumulation of DNA strand breaks that induce cell injury. For this reason, PARP-inhibitors (PARPi) have become promising tools to counteract many cancer types. One of the most used by clinicians is olaparib, that, however, showed again cancer resistance in patients. Thus, new generation molecules have been designed mainly to counteract this problem. Among them, we chose to test AZD2461 on the particularly aggressive human melanoma A375 cell line. This drug is a PARPi significantly less prone than olaparib to undergo the P-glycoprotein-mediated efflux mechanism, one of those responsible for resistance, that in turn is the main adversity in melanoma therapy. Then, we analysed AZD2461 also together with the enzyme onconase (ONC) on the same A375 cells, to investigate if the combination of drugs could possibly increase the in vitro antitumor activity. ONC is a small amphibian "pancreatic-type" ribonuclease that is able to exert a remarkable antitumor activity against many cancers, either in vitro or in vivo, principally because it can evade the ubiquitous ribonuclease cytosolic inhibitor thanks to its structural determinants. Hence, ONC became relevant in the use of protein-drug strategies against incurable cancers. The studies performed in this work showed that both drugs definitely affect A375 cells viability by inducing cytostatic and pro-apoptotic effects in a time- and dose-dependent manner, either if administered alone or in combination. Although we registered low synergistic effects with the combination of the two drugs, we found that AZD2461 did not induce resistance in A375 after two months treatment with high concentration of this molecule. Moreover, we underline that A375 cells treated for a prolonged time with AZD2461 were definitely more susceptible than parental A375 cells to the pro-apoptotic action of ONC. Considering also the different inhibitory effects of the two drugs on TNF-α gene expression and NF-kB DNA-binding, the tuning of their combined delivery to the A375 tumor cell line might open a promising scenario for future therapeutic applications devoted to defeat human melanoma
Onconase Restores Cytotoxicity in Dabrafenib-Resistant A375 Human Melanoma Cells and Affects Cell Migration, Invasion and Colony Formation Capability
Get PDFMelanoma is a lethal tumor because of its severe metastatic potential, and serine/threonine-protein kinase B-raf inhibitors (BRAFi) are used in patients harboring BRAF-mutation. Unfortunately, BRAFi induce resistance. Therefore, we tested the activity of onconase (ONC), a cytotoxic RNase variant, against BRAFi-resistant cells to re-establish the efficacy of the chemotherapy. To do so, an A375 dabrafenib-resistant (A375DR) melanoma cell subpopulation was selected and its behavior compared with that of parental (A375P) cells by crystal violet, 5-Bromo-2'-deoxyuridine incorporation, and cleaved poly(ADP-ribose) polymerase 1 (PARP1) western blot measurements. Then, nuclear p65 Nuclear Factor kappaB (NF-κB) and IκB kinases-α/β (IKK) phosphorylation levels were measured. Gelatin zymography was performed to evaluate metalloproteinase 2 (MMP2) activity. In addition, assays to measure migration, invasion and soft agar colony formation were performed to examine the tumor cell dissemination propensity. ONC affected the total viability and the proliferation rate of both A375P and A375DR cell subpopulations in a dose-dependent manner and also induced apoptotic cell death. Among its pleiotropic effects, ONC reduced nuclear p65 NF-κB amount and IKK phosphorylation level, as well as MMP2 activity in both cell subpopulations. ONC decreased cell colony formation, migration, and invasion capability. Notably, it induced apoptosis and inhibited colony formation and invasiveness more extensively in A375DR than in A375P cells. In conclusion, ONC successfully counteracts melanoma malignancy especially in BRAFi-resistant cells and could become a tool against melanoma recurrence
Onconase cytotoxicity in melanoma cancer cells: promising results to be enforced by oligomerizing the protein
No full textOnconase (ONC) is a 104 residues basic protein extracted from the Rana Pipiens frog oocytes which belongs to the “pancreatic-type” ribonuclease super-family. In fact, although RNase A and ONC share less than 30% sequence identity, they display similar folds. Contrarily to most monomeric RNases, ONC evades the cytosolic RNase inhibitor and exerts a remarkable cytotoxic activity against cancer cells1, which display more negatively membranes than normal cells. ONC cytotoxicity has already been tested against some cancer lines and exploited in clinical trials against non-small-lung cancer and unresectable malignant mesothelioma2. Human melanoma, an aggressive malignancy characterized by rapidly growing incidence and high mortality rate, is resistant to radiation therapy and cytotoxic chemotherapy3. Hence, we evaluated here the ONC effects against melanoma registering a remarkable cytotoxic effect in the A375 melanoma cell line, while negligible in NHEM melanocytes. We also investigated with WB some aspects underlying these encouraging results. Then, we recently found that ONC can dimerize through the 3D domain swapping mechanism and that its cytotoxicity increases against pancreatic cancer cells upon dimerization4. Hence, we are evaluating the ONC dimerization effect in A375 cells. Notably, native ONC can dimerize only through the swapping of its N-termini because its C-terminal ends are blocked by the C87-C104 disulfide. Considering that RNase C-swapped oligomers are generally more active than the N-swapped ones5, we are producing some ONC mutants devoted to enhance the oligomerization tendency of the protein. This should increase the ONC cytotoxic activity and contemporarily counteract undesired side-effects
Going Beyond Counting First Authors in Author Co-citation Analysis
Get PDFThe present study examines one of the fundamental aspects of author co-citation analysis (ACA) - the way co-citation
counts are defined. Co-citation counting provides the data on which all subsequent statistical analyses and mappings
are based, and we compare ACA results based on two different types of co-citation counting - the traditional type that
only counts the first one among a cited work's authors on the one hand and a non-traditional type that takes into
account the first 5 authors of a cited work on the other hand. Results indicate that the picture produced through this non-traditional author co-citation counting contains more coherent author groups and is therefore considerably clearer. However, this picture represents fewer specialties in the research field being studied than that produced through the traditional first-author co-citation counting when the same number of top-ranked authors is selected and analyzed. Reasons for these effects are discussed
Variations on the Author
Get PDF“Variations on the Author” discusses two of Eduardo Coutinho’s recent films (Um Dia na Vida, from 2010, and Últimas Conversas, posthumously released in 2015) and their contribution to the general question of documentary authorship. The director’s filmography is characterized by a consistent yet self-effacing form of authorial self-inscription: Coutinho often features as an interviewer that rather than express opinions propels discourses; an interviewer that is good at listening. This mode of self-inscription characterizes him as an author who is not expressive but who is nonetheless markedly present on the screen. In Um Dia na Vida, however, Coutinho is completely absent form the image, while Últimas Conversas, on the contrary, includes a confessional prologue that moves the director from the margins to the center of his films. This article examines the ways in which these works stand out in the filmography of a director who offers new insights into the notion of cinematic authorship
Appropriate Similarity Measures for Author Cocitation Analysis
Get PDFWe provide a number of new insights into the methodological discussion about author cocitation analysis. We first argue that the use of the Pearson correlation for measuring the similarity between authors’ cocitation profiles is not very satisfactory. We then discuss what kind of similarity measures may be used as an alternative to the Pearson correlation. We consider three similarity measures in particular. One is the well-known cosine. The other two similarity measures have not been used before in the bibliometric literature. Finally, we show by means of an example that our findings have a high practical relevance.information science;Pearson correlation;cosine;similarity measure;author cocitation analysis
Dispelling the Myths Behind First-author Citation Counts
Get PDFWe conducted a full-scale evaluative citation analysis study of scholars in the XML research field to explore just how different from each other author rankings resulting from different citation counting methods actually are, and to demonstrate the capability of emerging data and tools on the Web in supporting more realistic citation counting methods. Our results contest some common arguments for the continued
use of first-author citation counts in the evaluation of scholars, such as high correlations between author rankings by first-author citation counts and other citation
counting methods, and high costs of using more realistic citation counting methods that are not well-supported by the ISI databases. It is argued that increasingly available digital full text research papers make it possible for citation analysis studies to go beyond what the ISI databases have directly supported and to employ more
sophisticated methods
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