In vitro and in vivo evaluation of NCX 4040 cytotoxic activity in human colon cancer cell lines

BACKGROUND: Nitric oxide-releasing nonsteroidal antiinflammatory drugs (NO-NSAIDs) are reported to be safer than NSAIDs because of their lower gastric toxicity. We compared the effect of a novel NO-releasing derivate, NCX 4040, with that of aspirin and its denitrated analog, NCX 4042, in in vitro and in vivo human colon cancer models and investigated the mechanisms of action underlying its antitumor activity. METHODS: In vitro cytotoxicity was evaluated on a panel of colon cancer lines (LoVo, LoVo Dx, WiDr and LRWZ) by sulforhodamine B assay. Cell cycle perturbations and apoptosis were evaluated by flow cytometry. Protein expression was detected by Western blot. In the in vivo experiments, tumor-bearing mice were treated with NCX 4040, five times a week, for six consecutive weeks. RESULTS: In the in vitro studies, aspirin and NCX 4042 did not induce an effect on any of the cell lines, whereas NCX 4040 produced a marked cytostatic dose-related effect, indicating a pivotal role of the -NO(2 )group. Furthermore, in LoVo and LRWZ cell lines, we observed caspase-9 and -3-mediated apoptosis, whereas no apoptotic effect was observed after drug exposure in WiDr or LoVo Dx cell lines. In in vivo studies, both NCX 4040 and its parental compound were administered per os. NCX 4040 induced a 40% reduction in tumor weight. Conversely, aspirin did not influence tumor growth at all. CONCLUSIONS: NCX 4040, but not its parental compound, aspirin, showed an in vitro and in vivo antiproliferative activity, indicating its potential usefulness to treat colon cancer

Study of molecular mechanisms of pro-apoptotic activity of NCX 4040, a novel nitric oxide-releasing aspirin, in colon cancer cell lines

<p>Abstract</p> <p>Background</p> <p>Despite numerous studies aimed at verifying the antitumor activity of nitric oxide-releasing nonsteroidal antiflammatory drugs (NO-NSAIDs), little is known about the molecular targets responsible for their antineoplastic properties. In the present study, we investigated the mechanisms underlying the cytotoxicity of NCX 4040, a novel NO-aspirin with promising antineoplastic action, in <it>in vitro </it>human colon cancer models.</p> <p>Methods</p> <p>The effect on tumor growth was evaluated in four human colon cancer cell lines (LoVo, LRWZ, WiDr and LoVo Dx) by sulforhodamine B assay, oxidative stress by immunohistochemistry, apoptosis by laddering assay, mitochondrial membrane potential (ΔΨ_m) by flow cytometry, and apoptosis- and chemoresistance-related markers by western-blot and real-time method, respectively. Prostaglandin E₂levels were determined by ELISA.</p> <p>Results</p> <p>NCX 4040 produced a higher cytotoxic effect in all the cell lines than that produced by other NO donors tested. In particular, in LoVo and LRWZ cells, NCX 4040 induced a cytocidal effect and apoptosis through p53 and NAG-1 expression, an early ΔΨ_mcollapse, and a sequential release of cytoplasmatic cytochrome c and caspase -9 and -3 active forms. 8-hydroxyguanine lesions, indicative of oxidative stress, were also observed. Conversely, in WiDr line, the drug caused a cytocidal effect, albeit not through apoptosis, and a concomitant increase in COX-2 activity. In LoVo Dx line, characterized by high levels drug resistance and DNA repair-related markers, only a cytostatic effect was observed, again in concomitance with the increase in COX-2 enzyme activity.</p> <p>Conclusion</p> <p>This study highlights the multiplicity of mechanisms involved in sensitivity or resistance to NCX 4040 and could provide useful indications for tailored therapy by identifying potentially drug-responsive tumors.</p

La configuración de la identidad nacional en los orígenes de la democracia argentina: el yrigoyenismo

The present project’s objective is to contribute to the debate on the origins of Argentine democracy based on the analysis of the conformation of the national identity. This study is motivated by having perceived the persistent need to take hold of a “homogeneous” political subject, such as the Nation or the People to accomplish an idea of national integration. In this sense, we propose to approach the emergence of radicalism and the phenomenon of yrigoyenismo, as well as its ways of constructing political identity, emphasizing the use and tradition of its political vocabulary, rather than its mechanisms of political action or its party structure in government. On the other hand, we are interested in analysing tensions and filiations among the speeches from the liberal, democratic and nationalist traditions in this shared social, cultural and political scenario. Even though radicalism is born as a party with essentially impersonal principles, as it is displayed in its Organic Chart of 1890, for Yrigoyen radicalism was not a political party, but a movement: the Nation itself. From the beginning, this idea is linked to the understanding of politics as an apostolate. Yrigoyen appears as the restorative hero sent by Providence (Yrigoyen, My life and doctrine, 1923). The Manifesto of the Radical Civic Union to the people of the Republic of March 30, 1916 calls on all Argentines to fulfil the “sacred” civic duty, because the country requires “a deep renewal of their ethical values.” This idea of the apostle and the sacrifice is intimately connected with conceiving the Radical Civic Union as a movement, as “the civic religion of the nation where successive generations may go in search of noble inspirations”. The Cause against the Regime is that of the Nation itself. Yrigoyen assumes the christian image of the apostle, playing with Jesus’ figure - in a logic of messiah, prophet, but also of sacrifice and via crucis- as restorer of the moral and political order of the republic. However, does Yrigoyen give a personal sense to the feeling of civic religiosity expressed by radicalism? Or is this sentiment born with Yrigoyen’s system of leadership representations?El presente proyecto de investigación tiene por objetivo contribuir al debate sobre los orígenes de la democracia en la Argentina a partir del análisis de la conformación de la identidad nacional. El haber detectado la persistencia de la necesidad de asirse de un sujeto político “homogéneo”, como la nación o el pueblo, para lograr una idea de integración nacional lo ha motivado. En este sentido, nos proponemos abordar el surgimiento del radicalismo y el fenómeno del yrigoyenismo, así como sus modos de construcción de la identidad política, poniendo el acento en el uso y la tradición de su vocabulario político, más que en sus mecanismos de acción política y en la forma de su estructura partidaria en el gobierno. Por otro lado, nos interesa analizar las tensiones y filiaciones entre los discursos adherentes a la tradición liberal, la tradición democrática y los nacionalismos, respectivamente, puestos en valor en este escenario social, cultural y político compartido. Si bien el radicalismo nace como un partido de principios esencialmente impersonal, como recita su Carta Orgánica de 1890, para Yrigoyen el radicalismo no era un partido político, sino un movimiento: la nación misma. Desde el principio, esta idea está unida a la comprensión de la política como un apostolado. Yrigoyen aparece como el héroe restaurador enviado por la Providencia (Yrigoyen, Mi vida y mi doctrina, 1923). El Manifiesto de la Unión Cívica Radical al pueblo de la República del 30 de marzo de 1916 llama a todos los argentinos a cumplir con el “sagrado” deber cívico, porque el país requiere “una profunda renovación de sus valores éticos”. Esta idea del apóstol y del sacrificio está íntimamente conectada con concebir a la Unión Cívica Radical como un movimiento, como “la religión cívica de la nación adonde las generaciones sucesivas puedan acudir en busca de nobles inspiraciones”. La causa contra el régimen es la de la nación misma. Yrigoyen asume la imagen cristiana del apóstol, jugando con la figura de Jesús –en una lógica de mesías, profeta, pero también de sacrificio y vía crucis– como restaurador del orden moral y político de la república. Ahora bien, ¿Yrigoyen dona un sentido personal al sentimiento de religiosidad cívica expresado por el radicalismo? ¿O este sentimiento nace con el sistema de representaciones del liderazgo de Yrigoyen

Addition of 5-fluorouracil to doxorubicin-paclitaxel sequence increases caspase-dependent apoptosis in breast cancer cell lines

INTRODUCTION: The aim of the study was to evaluate the activity of a combination of doxorubicin (Dox), paclitaxel (Pacl) and 5-fluorouracil (5-FU), to define the most effective schedule, and to investigate the mechanisms of action in human breast cancer cells. METHODS: The study was performed on MCF-7 and BRC-230 cell lines. The cytotoxic activity was evaluated by sulphorhodamine B assay and the type of drug interaction was assessed by the median effect principle. Cell cycle perturbation and apoptosis were evaluated by flow cytometry, and apoptosis-related marker (p53, bcl-2, bax, p21), caspase and thymidylate synthase (TS) expression were assessed by western blot. RESULTS: 5-FU, used as a single agent, exerted a low cytotoxic activity in both cell lines. The Dox→Pacl sequence produced a synergistic cytocidal effect and enhanced the efficacy of subsequent exposure to 5-FU in both cell lines. Specifically, the Dox→Pacl sequence blocked cells in the G2-M phase, and the addition of 5-FU forced the cells to progress through the cell cycle or killed them. Furthermore, Dox→Pacl pretreatment produced a significant reduction in basal TS expression in both cell lines, probably favoring the increase in 5-FU activity. The sequence Dox→Pacl→48-h washout→5-FU produced a synergistic and highly schedule-dependent interaction (combination index < 1), resulting in an induction of apoptosis in both experimental models regardless of hormonal, p53, bcl-2 or bax status. Apoptosis in MCF-7 cells was induced through caspase-9 activation and anti-apoptosis-inducing factor hyperexpression. In the BRC-230 cell line, the apoptotic process was triggered only by a caspase-dependent mechanism. In particular, at the end of the three-drug treatment, caspase-8 activation triggered downstream executioner caspase-3 and, to a lesser degree, caspase-7. CONCLUSION: In our experimental models, characterized by different biomolecular profiles representing the different biology of human breast cancers, the schedule Dox→Pacl→48-h washout→5-FU was highly active and schedule-dependent and has recently been used to plan a phase I/II clinical protocol

Comparison of Coated and Immobilized Chiral Stationary Phases Based on Amylose tris-[(S)-α-Methylbenzylcarbamate] for the HPLC Enantiomer Separation of α-Lipoic Acid and Its Reduced Form

The couple of chiral sulfur compounds α-lipoic acid (ALA)/α-dihydrolipoic acid (DHALA) has attracted considerable attention in recent years owing to its remarkable anti-inflammatory and antioxidant properties. It is well known that the chirality of the C6 plays a key role in determining the biological activity of ALA. The natural occurring (R)-ALA enantiomer is an essential cofactor for key oxidative metabolism enzyme complexes and, after oral administration of the racemic mixture, it shows higher plasma concentration than (S)-ALA. Differently, the in vivo enantioselective action difference between the enantiomers of DHALA has not yet been studied. This lacking is perhaps due to the unavailability of analytical methods capable of determining the enantiomeric composition of biological samples during pharmacokinetic and pharmacodynamic events. In the present work, the direct and baseline enantioresolution of both chiral acids by HPLC on two amylose-derived chiral stationary phases is presented. The proposed chiral enantioselective protocol, therefore, does not require pre- or on-column derivatization. The performance of the coated Chiralpak AS-H CSP and the new immobilized Chiralpak IH-3 CSP, which have the same chiral selector amylose tris-[(S)-α-methylbenzylcarbamate], were compared using conventional normal-phase mobile phases containing ethanol or 2-propanol as alcoholic solvents and a fixed percentage of trifluoroacetic acid. Nonconventional eluents containing dichloromethane, ethyl acetate, and 2-methyltetrahydrofuran as organic cosolvents were applied in the separation of the enantiomers of two carboxylic acids on the immobilized Chiralpak IH-3 CSP. The effect of the column temperature was carefully evaluated in order to improve enantioselectivity. Adequate amounts of enantiomers were isolated by an analytical-size Chiralpak IH-3 column and submitted to chiroptical measurements. The absolute configuration assignment of the isolated enantiomers was determined by a multidisciplinary procedure based on the comparison of the experimental and calculated chiroptical properties