Heparanase and COX-2 expression as predictors of lymph node metastasis in large, high-grade breast tumors

Background/Aim: Heparanase (HPA) contributes to breast cancer metastasis by facilitating the breakdown of the basement membrane and extracellular matrix. High expression of HPA is thought to be associated with increased nodal involvement and poor survival in patients with breast cancer. Overexpression of cyclooxygenase-2 (COX-2) in breast cancer is associated with indicators of poor prognosis such as lymph node metastasis, poor differentiation, and large tumor size. The underlying mechanism by which HPA and COX-2 overexpression increases the metastatic potential of breast cancer is not fully-understood. To enhance our understanding over these mechanisms, we aimed to investigate the relationship between the size of the tumor and HPA expression, tumor grade as well as lymph node status in patients with breast cancer. Materials and Methods: Immunohistochemical analysis of HPA and COX-2 expression was performed on 246 breast tumor samples. The expression of HPA was correlated with COX-2 expression, tumor grade, lymph node status, oestrogen receptor status. Results: The overexpression of HPA and COX-2 was associated with increased likelihood of lymph node positivity in large, high-grade tumors. High-grade tumors with size greater than 20 mm, that overexpressed HPA, were 4-times more likely to be associated with lymph node involvement (OR 4.71, CI 1.21-18.25). Whereas, tumors greater than 20 mm in size were 5-times more likely to metastasize to the regional lymph nodes, if associated with overexpression of COX-2 (OR 5.5, CI 1.2-24.8). Conclusion: Expression of HPA appears to be a key mechanism by which large, highgrade breast tumors metastasize to regional lymph nodes, while COX-2 overexpression may be an independent predictor of lymph node positivity.

Clinical translation of a hand-held optical imager for breast cancer diagnostics: In vitro and in vivo tomography studies

Optical imaging is an emerging technology towards non-invasive breast cancer diagnostics. In recent years, portable and patient comfortable hand-held optical imagers are developed towards two-dimensional (2D) tumor detections. However, these imagers are not capable of three-dimensional (3D) tomography because they cannot register the positional information of the hand-held probe onto the imaged tissue. A hand-held optical imager has been developed in our Optical Imaging Laboratory with 3D tomography capabilities, as demonstrated from tissue phantom studies. The overall goal of my dissertation is towards the translation of our imager to the clinical setting for 3D tomographic imaging in human breast tissues. A systematic experimental approach was designed and executed as follows: (i) fast 2D imaging, (ii) coregistered imaging, and (iii) 3D tomographic imaging studies. (i) Fast 2D imaging was initially demonstrated in tissue phantoms (1% Liposyn solution) and in vitro (minced chicken breast and 1% Liposyn). A 0.45 cm3 fluorescent target at 1:0 contrast ratio was detectable up to 2.5 cm deep. Fast 2D imaging experiments performed in vivo with healthy female subjects also detected a 0.45 cm3 fluorescent target superficially placed ∼2.5 cm under the breast tissue. (ii) Coregistered imaging was automated and validated in phantoms with ∼0.19 cm error in the probe’s positional information. Coregistration also improved the target depth detection to 3.5 cm, from multi-location imaging approach. Coregistered imaging was further validated in-vivo , although the error in probe’s positional information increased to ∼0.9 cm (subject to soft tissue deformation and movement). (iii) Three-dimensional tomography studies were successfully demonstrated in vitro using 0.45 cm3 fluorescence targets. The feasibility of 3D tomography was demonstrated for the first time in breast tissues using the hand-held optical imager, wherein a 0.45 cm3 fluorescent target (superficially placed) was recovered along with artifacts. Diffuse optical imaging studies were performed in two breast cancer patients with invasive ductal carcinoma. The images showed greater absorption at the tumor cites (as observed from x-ray mammography, ultrasound, and/or MRI). In summary, my dissertation demonstrated the potential of a hand-held optical imager towards 2D breast tumor detection and 3D breast tomography, holding a promise for extensive clinical translational efforts.

Reduced Androgen Receptor Expression Accelerates the Onset of ERBB2 Induced Breast Tumors in Female Mice

Androgen receptor (AR) is commonly expressed in both the epithelium of normal mammary glands and in breast cancers. AR expression in breast cancers is independent of estrogen receptor alpha (ERα) status and is frequently associated with overexpression of the ERBB2 oncogene. AR signaling effects on breast cancer progression may depend on ERα and ERBB2 status. Up to 30% of human breast cancers are driven by overactive ERBB2 signaling and it is not clear whether AR expression affects any steps of tumor progression in this cohort of patients. To test this, we generated mammary specific Ar depleted mice (MARKO) by combining the floxed allele of Ar with the MMTV-cre transgene on an MMTV-NeuNT background and compared them to littermate MMTV-NeuNT, Arfl/+ control females. Heterozygous MARKO females displayed reduced levels of AR in mammary glands with mosaic AR expression in ductal epithelium. The loss of AR dramatically accelerated the onset of MMTV-NeuNT tumors in female MARKO mice. In this report we show that accelerated MMTV-NeuNT-dependent tumorigenesis is due specifically to the loss of AR, as hormonal levels, estrogen and progesterone receptors expression, and MMTV-NeuNT expression were similar between MARKO and control groups. MMTV-NeuNT induced tumors in both cohorts displayed distinct loss of AR in addition to ERα, PR, and the pioneer factor FOXA1. Erbb3 mRNA levels were significantly elevated in tumors in comparison to normal mammary glands. Thus the loss of AR in mouse mammary epithelium accelerates malignant transformation rather than the rate of tumorigenesis.

Fluorine-18-labeled estrogens, progestins and corticosteroids for receptor-based imaging of breast tumors and target areas of the brain

Estrogens can be labeled with the positron-emitting radionuclide fluorine-18 (t$\sb{1/2}$ = 110 min) by fluoride ion (n-Bu$\sb4$N$\sp{18}$F) displacement of a 16$\beta$-trifluoromethanesulfonate (triflate) derivative of the corresponding estrone 3-triflate, and purification by HPLC. That sequence has been used to synthesize the 11$\beta$-methoxy 1 and 11$\beta$-ethyl 2 analogues of the breast tumor imaging agent, 16$\alpha$-($\sp{18}$F) fluoro-17$\beta$-estradiol (FES). Tissue distribution studies of 1 and 2 in immature female rats show high selectivity for target tissue (T, uterus) vs non-target (NT, muscle and lung), with T/NT ratios being 43 and 17 at one hour after injection for 1 and 2, respectively. The parent estrogen FES has previously been shown to display an intermediate value for tissue selectivity.

Transcriptional pathway signatures predict MEK addiction and response to selumetinib (AZD6244)

Selumetinib (AZD6244, ARRY-142886) is a selective, non-ATP-competitive inhibitor of mitogen-activated protein/extracellular signal-regulated kinase kinase (MEK)-1/2. The range of antitumor activity seen preclinically and in patients highlights the importance of identifying determinants of response to this drug. In large tumor cell panels of diverse lineage, we show that MEK inhibitor response does not have an absolute correlation with mutational or phospho-protein markers of BRAF/MEK, RAS, or phosphoinositide 3-kinase (PI3K) activity. We aimed to enhance predictivity by measuring pathway output through coregulated gene networks displaying differential mRNA expression exclusive to resistant cell subsets and correlated to mutational or dynamic pathway activity. We discovered an 18-gene signature enabling measurement of MEK functional output independent of tumor genotype. Where the MEK pathway is activated but the cells remain resistant to selumetinib, we identified a 13-gene signature that implicates the existence of compensatory signaling from RAS effectors other than PI3K. The ability of these signatures to stratify samples according to functional activation of MEK and/or selumetinib sensitivity was shown in multiple independent melanoma, colon, breast, and lung tumor cell lines and in xenograft models. Furthermore, we were able to measure these signatures in fixed archival melanoma tumor samples using a single RT-qPCR-based test and found intergene correlations and associations with genetic markers of pathway activity to be preserved. These signatures offer useful tools for the study of MEK biology and clinical application of MEK inhibitors, and the novel approaches taken may benefit other targeted therapies.

Copper(I) complexes of modified nucleobases and vitamin B3 as potential chemotherapeutic agents: In vitro and in vivo studies

Three new complexes of Cu(I) have been synthesized using ancillary ligands like thiopyrimidine (tp) a modified nucleobase, and nicotinamide (nie) or vitamin B3, and characterized by spectroscopy and X-ray crystallography. In vitro cytotoxicity studies of the complexes on various human cancer cell lines such as Colo295, H226, HOP62, K562, MCF7 and T24 show that Cu(PPh3)(2)(tp)Cl] and Cu(PPh3)(2)(tp)ClO4 (2) have in vitro cytotoxicity comparable to cisplatin. Complex Cu(nic)(3)PPh3]ClO4 (3) is non-toxic and increases the life span by about 55 % in spontaneous breast tumor model. DNA binding and cleavage studies show that complex (3) binds to calf thymus DNA with an apparent binding constant of 5.9 x 10(5)M and completely cleaves super-coiled DNA at a concentration of 400 mu M, whereas complexes (1) and (2) do not bind DNA and do not show any cleavage even at 1200 mu M. Thus, complex (3) may exhibit cytotoxicity Via DNA cleavage whereas the mechanism of cytotoxicity of (1) and (2) probably involves a different pathway.

Estudo da cromatina nos sítios de fixação à matriz nuclear no domínio do gene TP53 e das modificações epigenéticas e no modelo de progressão tumoral mamária 21T

Dentre os diversos tipos de câncer agressivos, o câncer de mama é o mais comum em mulheres. Mutações hereditárias e adquiridas, assim como alterações epigenéticas atuam em sinergia na carcinogênese mamária e na progressão tumoral. A proteína P53 é uma supressora de tumor e possui uma atuação fundamental na integridade genômica. Apesar do vasto conhecimento sobre o controle da P53 a nível de proteína, ainda pouco se sabe sobre o controle transcricional do gene TP53. A série 21T, uma série de 4 linhagens celulares originadas da mama da mesma paciente, representando diferentes estágios de progressão tumoral mamária, é um eficiente modelo para investigação das alterações epigenéticas e suas influências na expressão gênica ao longo da progressão do câncer de mama. Nós analisamos a organização do domínio do gene TP53 através da técnica de arranjo de DNA, em diversas linhagens celulares de câncer de mama e linhagens controle, e realizamos uma tentativa de caracterizar estes elementos de DNA nas linhagens controle não-tumorais HB2 e MCF10A e nas tumorais MCF-7, MDA-MB-231, T47D, através dos marcadores epigenéticos de eucromatina, H4Ac, e heterocromatina, H3K9me3. Ainda analisamos a ligação de proteínas à região associada à matriz nuclear (MAR), denominada MAR 2, e a possível ligação da proteína ligante à matriz nuclear (MARBP), PARP-1, através de ensaios de gel shift (EMSA). Detectamos que na linhagem controle epitelial mamária, HB2, o gene TP53 está posicionado num domínio de DNA relativamente pequeno, aproximadamente 50 kb, delimitado por dois sítios de fixação à matriz nuclear. Interessantemente, esta estrutura de domínio se apresentou radicalmente diferente nas linhagens de câncer de mama estudadas, MCF7, T47D, MDA-MB-231 e BT474, nos quais o tamanho do domínio estudado estava aumentado e a transcrição do TP53 diminuída. Os enriquecimentos com os marcadores epigenéticos de cromatina H4Ac e H3K9me3 estão diferentemente distribuídos nas MARs nas linhagens celulares. Surpreendentemente, a MAR 2 apresentou uma ligação altamente específica, o que poderia representar a atuação de fatores transcricionais envolvidos na organização da cromatina. Através de programas de bioinformática, detectamos putativos sítios para interessantes fatores de transcrição, tais como o c/EBP-beta e c-myb, que poderiam atuar em cis regulando a expressão do gene TP53 e outros flanqueadores. Nós propusemos um modelo para a organização da cromatina na região de domínio do gene TP53 com os genes flanqueadores. Através da série 21T, detectamos uma hipometilação global genômica, nas células cancerosas 21NT e 21MT1. Uma importante diminuição da expressão global do marcador H4Ac nas células metastáticas 21MT1, foi detectada em relação às outras linhagens. Os níveis de RNAm das principais enzimas relacionadas as modificações epigenéticas são consistentes com as observadas hipometilação genômica e hipoacetilação. Através de microscopia confocal, verificamos que o marcador H4Ac está localizado, na maior parte na periferia e o marcador H3K9me3, pericêntrico nos núcleos tumorais. Por fim, verificamos que o promotor P1 do gene TP53 apresenta um estado de cromatina aberta, e a expressão do gene TP53 é similar em todas as células da série 21T.

Estudo preliminar da variação dos níveis de cortisol em doentes da espécie Canis familiaris sujeitos a mastectomia por doença oncológica da mama

Dissertação de Mestrado Integrado em Medicina Veterinária

Ataxia telangiectasia mutated (ATM) inhibition transforms human mammary gland epithelial cells.

Carriers of mutations in the cell cycle checkpoint protein kinase ataxia telangiectasia mutated (ATM), which represent 1-2% of the general population, have an increased risk of breast cancer. However, experimental evidence that ATM deficiency contributes to human breast carcinogenesis is lacking. We report here that in MCF-10A and MCF-12A cells, which are well established normal human mammary gland epithelial cell models, partial or almost complete stable ATM silencing or pharmacological inhibition resulted in cellular transformation, genomic instability, and formation of dysplastic lesions in NOD/SCID mice. These effects did not require the activity of exogenous DNA-damaging agents and were preceded by an unsuspected and striking increase in cell proliferation also observed in primary human mammary gland epithelial cells. Increased proliferation correlated with a dramatic, transient, and proteasome-dependent reduction of p21(WAF1/CIP1) and p27(KIP1) protein levels, whereas little or no effect was observed on p21(WAF1/CIP1) or p27(KIP1) mRNAs. p21(WAF1/CIP1) silencing also increased MCF-10A cell proliferation, thus identifying p21(WAF1/CIP1) down-regulation as a mediator of the proliferative effect of ATM inhibition. Our findings provide the first experimental evidence that ATM is a human breast tumor suppressor. In addition, they mirror the sensitivity of ATM tumor suppressor function and unveil a new mechanism by which ATM might prevent human breast tumorigenesis, namely a direct inhibitory effect on the basal proliferation of normal mammary epithelial cells.

Régulation de l’activité transcriptionnelle des récepteurs des estrogènes (ER) par le récepteur à chimiokine CXCR4 et les récepteurs à activité tyrosine kinase ErbB2 et ErbB3

La régulation de la transcription des gènes par les récepteurs des estrogènes ERα et ERβ joue un rôle important dans la croissance cellulaire et dans le développement du cancer du sein. Une augmentation de l’expression de CXCR4 et de son ligand SDF-1/CXCL12 corrèle avec un phénotype plus agressif du cancer du sein. Ici, nous démontrons un mécanisme de boucle de régulation positive entre la signalisation de CXCR4/SDF-1 et l’activité transcriptionnelle des ERs dans des cellules cancéreuses mammaires. L’activité transcriptionnelle de ER et l’expression de gènes cibles de ER, dont SDF-1 lui-même, sont augmentées dans la lignée cancéreuse mammaire MCF-7 en réponse à SDF-1. Ces effets sont bloqués par l’anti-estrogène fulvestrant et par la délétion de CXCR4. Par ailleurs, l’expression des gènes et la prolifération des cellules cancéreuses mammaires MCF-7 en réponse à l’estrogène sont altérées par l’inhibition de CXCR4. La signalisation par les facteurs de croissance joue un rôle important dans le cancer du sein. La surexpression et la dérégulation de la signalisation par le récepteur à activité tyrosine kinase ErbB2 corrèlent avec un phénotype tumoral mammaire plus agressif et un moins bon pronostic. Cependant, comment la signalisation de ErbB2 et de CXCR4 sont fonctionnellement reliées dans la régulation de la réponse de ER dans les cellules cancéreuses mammaires n’est pas connue. Nous démontrons ici que CXCR4 régule négativement l’expression protéique de ErbB2 et de son partenaire d’interaction ErbB3 ainsi que la phosphorylation de ErbB2. CXCR4 altère l’activation de la voie PI3-K/Akt par le dimère ErbB2/ErbB3 en réponse à héréguline alors qu’en présence de SDF-1, les niveaux d’activation sont récupérés. Nous avons trouvé que héréguline-β promouvoit la phosphorylation de la sérine 339 de CXCR4, un site important pour l’internalisation et la signalisation du récepteur. De plus, le recrutement de ErbB2 à CXCR4 est favorisé par ErbB3 et héréguline-β. L’activité transcriptionnelle ainsi que l’expression des gènes cibles de ER en réponse à l’héréguline sont relevées avec l’expression de CXCR4 et partiellement récupérées avec l’addition de SDF-1. Ces résultats démontrent que le recrutement de CXCR4 à ErbB2 altère la signalisation médiée par ErbB2/ErbB3 ainsi que l’activité hormonale de ER dans des cellules cancéreuses mammaires. Nous travaux ont permis d’identifier et de caractériser l’impact de la signalisation médiée par des récepteurs membranaires sur la réponse transcriptionnelle de ER dans des cellules cancéreuses mammaires. La signalisation membranaire est un facteur pouvant contribuer à la résistance aux thérapies endocriniennes et donc cibler les récepteurs impliqués s’avèrerait utile pour améliorer les traitements existants et mettre au point de nouvelles approches.

Determinación de anomalías cromosómicas y secuencias de ADN amplificadas en cáncer de mama

El cáncer de mama en Colombia, es la tercera causa de muerte en la población en general y la segunda en mujeres. En el año 2002 el 40.5% de los casos se presentaron en mujeres menores de 50 años (Pardo, et al. 2003). El cáncer de mama resulta de múltiples factores, entre los que se incluyen cambios sucesivos en el genoma de células epiteliales originalmente normales, que pueden conducir a la activación de oncogenes, inactivación de genes supresores de tumor y pérdida de función de genes reparadores de daños al ADN. Estas alteraciones pueden también ser producto de anomalías cromosómicas tales como monosomías, trisomías, translocaciones, inversiones, pérdida de material genético y amplificaciones que también afectan la expresión de genes (1) (2) (3) (4). Sin embargo, el orden de aparición de los diferentes eventos no está completamente dilucidado. En este estudio se determinaron las anomalías cromosómicas y secuencias de ADN amplificadas en pacientes con cáncer de mama, tanto en muestras de sangre periférica como de tumor de mama de 30 pacientes. En las dos líneas celulares analizadas se observó una alta frecuencia de monosomías principalmente de los cromosomas X, 6, 7, 9, 17, 19 y 22. Hay una asociación entre las monosomías de los cromosomas 17 y 22 con el estado negativo para los receptores de estrógenos y progestágenos (p=0.027, p=0.050). También se encontró asociación entre la monosomía del cromosoma 19 con edad avanzada (p=0.034), observándose formas más agresivas de la enfermedad cuando ésta estuvo presente. Las monosomías fueron características de carcinomas ductales infiltrantes de todos los grados. En los demás tipos de carcinoma su frecuencia fue más baja. En el presente estudio se encontró una asociación significativa entre algunas anomalías cromosómicas y la enfermedad, no reportadas anteriormente, como fueron algunas monosomías, fragilidades y roturas cromosómicas y cromatídicas. La alta frecuencia de fragilidades encontradas tanto en sangre periférica (fra 9q12 p=0.001 y fra 3p14 p= 0.38) como de fragilidades expresadas espontáneamente (no inducidas por el uso de reactivos específicos) en muestras de tumor de mama (fra 1p11 p= 0.001, fra 2q11 p= 0.002), pueden ser el reflejo de una alta inestabilidad cromosómica en el genoma de estos pacientes, mostrando lautilidad de los estudios de fragilidad en la determinación de individuos en alto riesgo de desarrollar cáncer de mama. En ensayos de FISH no se observaron amplificaciones de los genes ERBb2 y c-myc en los pacientes analizados. Esto concuerda con lo encontrado en la literatura en donde se ha reportado, para este tipo de tumores, una sobre expresión de la proteína sin amplificación del gen, explicada por desregulación de la expresión del gen, a su vez posiblemente debida a mutaciones en la región promotora o a alteraciones, que conducen a un aumento de la tasa de transcripción (5) (6) (7). Los resultados obtenidos, aunque preliminares, aportan nuevos marcadores cromosómicos que pueden orientar el diagnóstico, pronóstico y tratamiento de esta patología.

Factores asociados a recaída en pacientes con cáncer de mama de una institución de Bogotá

Introducción: El cáncer de seno es la primera causa de cáncer entre las mujeres, además es la primera causa de muerte por cáncer entre las hispanas y la segunda entre otras razas, sin contar con el gran impacto social y económico que conlleva esta patología. Esto motiva la realización de estudios propios, que permitan ampliar nuestro conocimiento y aportar a la literatura colombiana, una publicación que refleje los factores asociados a la recaída en el cáncer de mama. Métodos: Estudio observacional analítico retrospectivo de casos y controles en el que se tomaron 267 historias clínicas de pacientes con diagnóstico de cáncer de seno, clasificadas según estadio clínico y expresión molecular del tumor, se analizaron los factores más fuertemente asociados a la recaída. Resultados: La población total consistió en 267 mujeres de las cuales 58 presentaron recaída, con un relación caso – control, 1:3. Al evaluar los grupos se evidencia homogeneidad en cuanto a edad, tipo de neoplasia, paridad e histología con lo que concluimos que estos grupos son comparables. Se presentó una tasa de mortalidad de 13,8 % en las pacientes que presentaron recaída tumoral vs un 0% de mortalidad en aquellas pacientes sin recaída. Adicionalmente se evidencia una relación entre la presencia del receptor HER 2 y recaída tumoral, que aunque no es estadísticamente significativa (p = 0.112) es importante tener en cuenta por su significancia clínica. Por su parte la presencia de receptor de estrógenos y progestágenos no es un predictor de recaída. La realización de cirugía se muestra como un factor de protección (OAR: 0.046 p = 0.008). Finalmente se encontró una asociación estadísticamente significativa como variables de asociación a recaída tumoral: la edad (p=0.009), el estadio clínico en el momento del diagnóstico (p= <0.001) y la clasificación molecular del tumor (p= 0.016). Conclusiones: Se identificaron como factores asociados a recaída tumoral en pacientes con cáncer de mama de una institución de Bogotá, Colombia a: la edad, el estadio clínico en el momento del diagnóstico y la clasificación molecular del tumor, confirmando la agresividad de los tumores triple negativos. Todos los hallazgos son compatibles a lo descrito en la literatura mundial. Esto permite definir la necesidad de generar en nuestro país estrategias de salud pública, que permitan la educación a todos los grupos etarios para el tamizaje en población joven que está siendo afectada, la detección en estadios tempranos del cáncer de mama, asociados a priorización del manejo y mejoras en la ruta de atención de las pacientes que permitan impactar positivamente en el desenlace y calidad de vida de las mujeres con esta patología. Adicionalmente estos resultados impulsan a la continua investigación de nuevas tecnologías y medicamentos que permitan combatir los tumores más agresivos molecularmente hablando.

Synthesis, characterization, X-ray structure and in vitro anti mycobacterial and antitumoral activities of Ru(II) phosphine/diimine complexes containing the ""SpymMe(2)"" ligand, SpymMe(2)=4,6-dimethyl-2-mercaptopyrimidine

The reaction of cis-[RuCl2(dppb)(N-N)], dppb = 1,4-bis(diphenylphosphino)butane, complexes with the ligand HSpymMe(2), 4,6-dimethyl-2-mercaptopyrimidine, yielded the cationic complexes [Ru(SpymMe(2))(dppb)(N-N)]PF6, N-N = bipy (1) and Me-bipy (2), bipy = 2,2`-bipyridine and Me-bipy = 4,4`dimethyl-2,2`-bipyridine, which were characterized by spectroscopic and electrochemical techniques and X-ray crystallography and elemental analysis. Additionally, preliminary in vitro tests for antimycobacterial activity against Mycobacterium tuberculosis H37Rv ATCC 27264 and antitumor activity against the MDA-MB-231 human breast tumor cell line were carried out on the new complexes and also on the precursors cis-[RuCl2(dppb)(N-N)], N-N = bipy (3) and Me-bipy (4) and the free ligands dppb, bipy, Me-bipy and SpymMe(2). The minimal inhibitory concentration (MIC) of compounds needed to kill 90% of mycobacterial cells and the IC50 values for the antitumor activity were determined. Compounds 1-4 exhibited good in vitro activity against M. tuberculosis, with MIC values ranging between 0.78 and 6.25 mu g/mL, compared to the free ligands (MIC of 25 to >50 mu g/mL) and the drugs used to treat tuberculosis. Complexes I and 2 also showed promising antitumor activity, with IC50 values of 0.46 +/- 0.02 and 0.43 +/- 0.08 mu M, respectively, against MDA-MB-231 breast tumor cells. (C) 2008 Elsevier Inc. All rights reserved.

Synthesis, characterization and cytotoxic activities of the [RuCl(2)(NO)(dppp)(L)]PF(6) complexes

The synthesis and characterization of ruthenium compounds of the type [RuCl(2)(NO)(dppp)(L)]PF(6) [dppp = 1,3-bis(diphenylphosphino)propane; L = pyridine, 4-methylpyridine, 4-phenylpyridine and dimethyl sulfoxide] are described. The complexes were characterized by elemental analysis, UV/Vis and infrared spectroscopy, cyclic voltammetry, and X-ray crystallography for the complexes with the pyridine and 4-methylpyridine ligands. In vitro evaluation of these nitrosyl complexes revealed cytotoxic activity from 7.1 to 19.0 mu M against the MDA-MB-231 breast tumor cells and showed that, in this case, they are more active than the reference metallodrug cisplatin. The 1,3-bis(diphenylphosphino)propane and the N-heterocyclic ligands alone failed to show cytotoxic activities at the concentrations tested (maximum concentration utilized = 200 mu M). (C) 2009 Elsevier Inc. All rights reserved.

Upregulation of glutathione-related genes and enzyme activities in cultured human cells by sublethgal concentrations of inorganic arsenic

Inorganic arsenic (jAs), a known human carcinogen, acts as a tumor promoter in part by inducing a rapid burst of reactive oxygen species (ROS) in mammalian cells. This causes oxidative stress and a subsequent increase in the level of cellular glutathione (GSH). Glutathione, a ubiquitous reducing sulfhydryl tripeptide, is involved in ROS detoxification and its increase may be part of an adaptive response to the oxidative stress. Glutathione related enzymes including glutathione reductase (GR) and glutathione S-transferase (GST) also play key roles in these processes. In this study the regulatory effects of inorganic arsenite (As111) on the activities of GSH-related enzymes were investigated in cultured human keratinocytes. Substantial increases in GR enzyme activity and mRNA levels were shown in keratinocytes and other human cell lines after exposure to low, subtoxic, micromolar concentrations of As111 for 24 h. Upregulation of GSH synthesis paralleled the upregulation of GR as shown by increases in glutamatecysteine lyase (GeL) enzyme activity and mRNA levels, cystine uptake, and intracellular GSH levels. Glutathione S-transferase activity was also shown to increase slightly in keratinocytes, but not in fibroblasts or breast tumor cells. Overall the results show that sublethal arsenic induces a multicomponent response in human keratinocytes that involves upregulation of parts, but not all of the GSH system and counteracts the acute toxic effects of jAs. The upregulation of GR has not previously been shown to be an integral part of this response, although GR is critical for maintaining levels of reduced GSH.