Synthesis of organometallic Ruthenium(II) complexes with strong activity against several human canceer cell lines

A new family of "RuCp" (Cp=eta(5)-C5H5) derivatives with bidentate N,O and N,N'-heteroaromatic ligands revealed outstanding cytotoxic properties against several human cell lines namely, A2780, A2780CisR, HT29, MCF7, MDAMB231, and PD. IC50 values were much lower than those found for cisplatin. Crystal structure of compound 4 was determined by X-ray diffraction studies. Density functional theory (DFT) calculations performed for compound 1 showed electronic flow from the ruthenium center to the coordinated bidentate ligand, in agreement with the electrochemical studies and the existence of a metal-to-ligand charge-transfer (MLCT) band evidenced by spectroscopic data.

Cobalt and Zinc Compounds Bearing 1,10-Phenanthroline-5,6-dione or 1,3,5-Triaza-7-phosphaadamantane Derivatives - Synthesis, Characterization, Cytotoxicity, and Cell Selectivity Studies

The compounds [mPTA][CoCl4] (1, mPTA = N-methyl-1,3,5-triaza-7-phosphaadamantane cation), [CoCl(H2O)(DION)(2)][BF4] (2, DION = 1,10-phenanthroline-5,6-dione), [Zn(DION)(2)]Cl-2 (3) and [ZnCl(O-PTA=O)(DION)][BF4] (4) were synthesized by reaction of CoCl2 with [mPTA]I or DION and ZnCl2 with DION or 1,3,5-triaza-7-phosphaadamantane-7-oxide (PTA=O) and DION, respectively. All complexes are water soluble and have been characterized by IR, far-IR, H-1, C-13 and P-31{H-1} NMR spectroscopy, ESI-MS, elemental analyses and single-crystal X-ray diffraction structural analysis (for 1). They were screened against the human tumour cell lines HCT116, HepG2 and MCF7. Complexes 2 and 3 exhibit the highest in vitro cytotoxicity and show lower cytotoxic activities in normal human fibroblast cell line than in HCT116 tumour cell line, which demonstrates their slight specificity for this type of tumour cell.

Tubulin cofactor A gene silencing in mammalian cells induces changes in microtubule cytoskeleton, cell cycle arrest and cell death

Microtubules are polymers of alpha/beta-tubulin participating in essential cell functions. A multistep process involving distinct molecular chaperones and cofactors produces new tubulin heterodimers competent to polymerise. In vitro cofactor A (TBCA) interacts with beta-tubulin in a quasi-native state behaving as a molecular chaperone. We have used siRNA to silence TBCA expression in HeLa and MCF-7 mammalian cell lines. TBCA is essential for cell viability and its knockdown produces a decrease in the amount of soluble tubulin, modifications in microtubules and G1 cell cycle arrest. In MCF-7 cells, cell death was preceded by a change in cell shape resembling differentiation.

Molecular fingerprint of human gastric cell line infected by Helicobacter pylori

Helicobacter pylori infection represents a serious health problem, given its association with serious gastric diseases as gastric ulcers, cancer and MALT lymphoma. Currently no vaccine exists and antibiotic-based eradication therapy is already failing in more than 20% of cases. To increase the knowledge on the infection process diverse gastric cell lines, e.g. the adenocarcinona gastric (AGS) cell line, are routinely used has in vitro models of gastric epithelia. In the present work the molecular fingerprint of infected and non-infected AGS cell lines, by diverse H. pylori strains, was acquired using vibrational infrared spectroscopy. These molecular fingerprints enabled to discriminate infected from non-infected AGS cells, and infection due to different strains, by performing Principal Component Analysis. It was also possible to estimate, from the AGS cells molecular fingerprint, the effect of the infection on diverse biochemical and metabolic cellular status. In resume infra-red spectroscopy enabled the acquisition of infected AGS cells molecular fingerprint with minimal sample preparation, in a rapid, high-throughput, economic process yielding highly sensitive and informative data, most useful for promoting critical knowledge on the H. pylori infection process. © 2015 IEEE.

Syntheses, Molecular Structures, Electrochemical Behavior, Theoretical Study, and Antitumor Activities of Organotin(IV) Complexes Containing 1-(4-Chlorophenyl)-1-cyclopentanecarboxylato Ligands

The organotin(IV) compounds [Me2Sn(L)(2)] (1), [Et(2)sn(L)(2)] (2), [(Bu2Sn)-Bu-n(L)(2)] (3), [(n)Oct(2)Sn(L)(2)] (4), [Ph2Sn(L)(2)] (5), and [PhOSnL](6) (6) have been synthesized from the reactions of 1-(4-chlorophenyl)-1-cyclopentanecarboxylic acid (HL) with the corresponding diorganotin(IV) oxide or dichloride. They were characterized by IR and multinuclear NMR spectroscopies, elemental analysis, cyclic voltammetry, and, for 2, 3, 4 and 6, single crystal X-ray diffraction analysis. While 1-5 are mononuclear diorganotin (IV) compounds, the X-ray diffraction of 6 discloses a hexameric drumlike structure with a prismatic Sn6O6 core. All these complexes undergo irreversible reductions and were screened for their in vitro antitumor activities toward HL-60, BGC-823, Bel-7402, and KB human cancer cell lines. Within the mononuclear compounds, the most active ones (3, 5) are easiest to reduce (least cathodic reduction potentials), while the least active ones (1, 4) are the most difficult to reduce. Structural rearrangements (i.e., Sn-O bond cleavages and trans-to-cis isomerization) induced by reduction, which eventually can favor the bioactivity, are disclosed by theoretical/electrochemical studies.

Alginate-chitosan particulate delivery systems for mucosal immunization against tuberculosis

Although vaccination is still the most cost-effective strategy for tuberculosis control, there is an urgent need for an improved vaccine. Current BCG vaccine lacks efficacy in preventing adult pulmonary tuberculosis, the most prevalent form of the disease. Targeting nasal mucosa, Mycobacterium tuberculosis infection site, will allow a simpler, less prone to risk of infection and more effective immunization against disease. Due to its biodegradable, immunogenic and mucoadhesive properties, chitosan particulate delivery systems can act both as carrier and as adjuvant, improving the elicited immune response. In this study, BCG was encapsulated in alginate and chitosan microparticles, via a mild ionotropic gelation procedure with sodium tripolyphosphate as a counterion. The particulate system developed shows effective modulation of BCG surface physicochemical properties, suitable for mucosal immunization. Intracellular uptake was confirmed by effective transfection of human macrophage cell lines.

Análogos do neuropéptido Y marcados com 99mTc para detecção de receptores Y1 expressos no cancro da mama

Mestrado em Medicina Nuclear. Área de especialização: Radiofarmácia.

Chitosan-alginate microparticulate delivery system for an alternative route of administration of BCG vaccine

Immunisation against M. tuberculosis with current available BCG vaccine lacks efficacy in preventing adult pulmonary tuberculosis. Targeting nasal mucosa is an attractive option for a more effective immunization. The delivery of BCG via the intranasal route involves overcoming barriers such as crossing the physical barrier imposed by the mucus layer and ciliar remotion, cellular uptake and intracellular trafficking by antigen presenting cells. Due to its biodegradable, immunogenic and mucoadhesive properties, chitosan particulate delivery systems can act both as vaccine carrier and adjuvant, improving the elicited immune response. In this study, different combinations of Chitosan/Alginate/TPP microparticles with BCG were produced as vaccine systems. The developed microparticle system successfully modulates BCG surface physicochemical properties and promotes effective intracellular uptake by human macrophage cell lines Preliminary immune responses were evaluated after s.c. and intranasal immunisation of BALB/c mice. BCG vaccination successfully stimulated the segregation of IgG2a and IgG1, where intranasal immunisation with chitosan/alginate particulate system efficiently elicited a more equilibrated cellular/humoral immune response.

In vivo and in vitro effects of RAD001 on bladder cancer

Objective: To evaluate the influence of Everolimus (RAD001) on chemically induced urothelial lesions in mice and its influence on in vitro human bladder cancer cell lines. Methods: ICR male mice were given N-butyl-N-(4-hydroxybutyl) nitrosamine in drinking water for a period of 12 weeks. Subsequently, RAD001 was administered via oral gavage, for 6 weeks. At the end of the experiment, all the animals were sacrificed and tumor development was determined by means of histopathologic evaluation; mammalian target of rapamycin (mTOR) expressivity was evaluated by immunohistochemistry. Three human bladder cancer cell lines (T24, HT1376, and 5637) were treated using a range of RAD001 concentrations. MTT assay, terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL), and flow cytometry were used to assess cell proliferation, apoptosis index, and cell cycle analysis, respectively. Immunoblotting analysis of 3 cell line extracts using mTOR and Akt antibodies was performed in order to study the expression of Akt and mTOR proteins and their phosphorylated forms. Results: The incidence of urothelial lesions in animals treated with RAD001 was similar to those animals not treated. RAD001 did not block T24 and HT1376 cell proliferation or induce apoptosis. A reduction in cell proliferation rate and therefore G0/G1 phase arrest, as well as a statistically significant induction of apoptosis (P 0.001), was only observed in the 5637 cell line. Conclusion: RAD001 seems not to have a significant effect on chemically induced murine bladder tumors. The effect of RAD001 on tumor proliferation and apoptosis was achieved only in superficial derived bladder cancer cell line, no effect was observed in invasive cell lines.

Biological activity and cellular uptake of [Ru(eta(5)-C5H5)(PPh3)(Me(2)bpy)][CF3SO3] complex

Anticancer activity of the new [Ru(eta(5)-C5H5)(PPh3)(Me(2)bpy)][CF3SO3] (Me(2)bpy = 4,4'-dimethyl-2,2'-bipyridine) complex was evaluated in vitro against several human cancer cell lines, namely A2780, A2780CisR, HT29, MCF7, MDAMB231 and PC3. Remarkably, the IC50 values, placed in the nanomolar and sub-micromolar range, largely exceeded the activity of cisplatin. Binding to human serum albumin, either HSA (human serum albumin) or HSA(faf) (fatty acid-free human serum albumin) does not affect the complex activity. Fluorescence studies revealed that the present ruthenium complex strongly quench the intrinsic fluorescence of albumin. Cell death by the [Ru(eta(5)-C5H5)(PPh3)(Me(2)bpy)][CF3SO3] complex was reduced in the presence of endocytosis modulators and at low temperature, suggesting an energy-dependent mechanism consistent with endocytosis. On the whole, the biological activity evaluated herein suggests that the complex could be a promising anticancer agent. (C) 2013 Elsevier Inc. All rights reserved.

Differential expression of CDC25 phosphatases splice variants in human breast cancer cells

Background: CDC25 phosphatases control cell cycle progression by activating cyclin dependent kinases. The three CDC25 isoforms encoding genes are submitted to alternative splicing events which generate at least two variants for CDC25A and five for both CDC25B and CDC25C. An over-expression of CDC25 was reported in several types of cancer, including breast cancer, and is often associated with a poor prognosis. Nevertheless, most of the previous studies did not address the expression of CDC25 splice variants. Here, we evaluated CDC25 spliced transcripts expression in anti-cancerous drug-sensitive and resistant breast cancer cell lines in order to identify potential breast cancer biomarkers. Methods: CDC25 splice variants mRNA levels were evaluated by semi-quantitative RT-PCR and by an original real-time RT-PCR assay. Results: CDC25 spliced transcripts are differentially expres-sed in the breast cancer cell lines studied. An up-regulation of CDC25A2 variant and an increase of the CDC25C5/C1 ratio are associated to the multidrug-resistance in VCREMS and DOXOR breast cancer cells, compared to their sensitive counterpart cell line MCF-7. Additionally, CDC25B2 tran-script is exclusively over-expressed in VCREMS resistant cells and could therefore be involved in the development of certain type of drug resistance. Conclusions: CDC25 splice variants could represent interesting potential breast cancer prognostic biomarkers.

Differential expression of GSPT1 GGCn alleles in cancer

The human eukaryotic release factor 3a (eRF3a), encoded by the G1 to S phase transition 1 gene (GSPT1; alias eRF3a), is upregulated in various human cancers. GSPT1 contains a GGCn polymorphism in exon 1, encoding a polyglycine expansion in the N-terminal of the protein. The longer allele, GGC12, was previously shown to be associated to cancer. The GGC12 allele was present in 2.2% of colorectal cancer patients but was absent in Crohn disease patients and in the control group. Real-time quantitative RT-PCR analysis showed that the GGC12 allele was present at up to 10-fold higher transcription levels than the GGC10 allele (P < 0.001). No GSPT1 amplifications were detected, and there was no correlation between the length of the alleles and methylation levels of the CpG sites inside the GGC expansion. Using flow cytometry, we compared the levels of apoptosis and proliferation rates between cell lines with different genotypes, but detected no significant differences. Finally, we used a cytokinesis-block micronucleus assay to evaluate the frequency of micronuclei in the same cell lines. Cell lines with the longer alleles had higher frequencies of micronuclei in binucleated cells, which is probably a result of defects in mitotic spindle formation. Altogether, these findings indicate that GSPT1 should be considered a potential proto-oncogene.

Cobalt complexes with pyrazole ligands as catalyst precursors for the peroxidative oxidation of cyclohexane: Z-ray absorption spectroscopy studies and biological applications

[CoCl(-Cl)(Hpz(Ph))(3)](2) (1) and [CoCl2(Hpz(Ph))(4)] (2) were obtained by reaction of CoCl2 with HC(pz(Ph))(3) and Hpz(Ph), respectively (Hpz(Ph)=3-phenylpyrazole). The compounds were isolated as air-stable solids and fully characterized by IR and far-IR spectroscopy, MS(ESI+/-), elemental analysis, cyclic voltammetry (CV), controlled potential electrolysis, and single-crystal X-ray diffraction. Electrochemical studies showed that 1 and 2 undergo single-electron irreversible (CoCoIII)-Co-II oxidations and (CoCoI)-Co-II reductions at potentials measured by CV, which also allowed, in the case of dinuclear complex 1, the detection of electronic communication between the Co centers through the chloride bridging ligands. The electrochemical behavior of models of 1 and 2 were also investigated by density functional theory (DFT) methods, which indicated that the vertical oxidation of 1 and 2 (that before structural relaxation) affects mostly the chloride and pyrazolyl ligands, whereas adiabatic oxidation (that after the geometry relaxation) and reduction are mostly metal centered. Compounds 1 and 2 and, for comparative purposes, other related scorpionate and pyrazole cobalt complexes, exhibit catalytic activity for the peroxidative oxidation of cyclohexane to cyclohexanol and cyclohexanone under mild conditions (room temperature, aqueous H2O2). Insitu X-ray absorption spectroscopy studies indicated that the species derived from complexes 1 and 2 during the oxidation of cyclohexane (i.e., Ox-1 and Ox-2, respectively) are analogous and contain a Co-III site. Complex 2 showed low invitro cytotoxicity toward the HCT116 colorectal carcinoma and MCF7 breast adenocarcinoma cell lines.

Insights into the mechanims underlyng the antiproliferative potential of a Co(II) coordination compound bearing 1,10-Phenanthroline-5,6-Dione: DNA and protein interaction studies

The very high antiproliferative activity of [Co(Cl)(H2O)(phendione)(2)][BF4] (phendione is 1,10-phenanthroline-5,6-dione) against three human tumor cell lines (half-maximal inhibitory concentration below 1 mu M) and its slight selectivity for the colorectal tumor cell line compared with healthy human fibroblasts led us to explore the mechanisms of action underlying this promising antitumor potential. As previously shown by our group, this complex induces cell cycle arrest in S phase and subsequent cell death by apoptosis and it also reduces the expression of proteins typically upregulated in tumors. In the present work, we demonstrate that [Co(Cl)(phendione)(2)(H2O)][BF4] (1) does not reduce the viability of nontumorigenic breast epithelial cells by more than 85 % at 1 mu M, (2) promotes the upregulation of proapoptotic Bax and cell-cycle-related p21, and (3) induces release of lactate dehydrogenase, which is partially reversed by ursodeoxycholic acid. DNA interaction studies were performed to uncover the genotoxicity of the complex and demonstrate that even though it displays K (b) (+/- A standard error of the mean) of (3.48 +/- A 0.03) x 10(5) M-1 and is able to produce double-strand breaks in a concentration-dependent manner, it does not exert any clastogenic effect ex vivo, ruling out DNA as a major cellular target for the complex. Steady-state and time-resolved fluorescence spectroscopy studies are indicative of a strong and specific interaction of the complex with human serum albumin, involving one binding site, at a distance of approximately 1.5 nm for the Trp214 indole side chain with log K (b) similar to 4.7, thus suggesting that this complex can be efficiently transported by albumin in the blood plasma.

New water-soluble Ruthenium(II) cytotoxic complex: biological activity and celular distribution

A novel water soluble organometallic compound, [RuCp(mTPPMSNa)(2,2'-bipy)][CF3SO3] (TM85, where Cp=eta(5)-cyclopentadienyl, mTPPMS = diphenylphosphane-benzene-3-sulfonate and 2,2'-bipy = 2,2'-bipyridine) is presented herein. Studies of interactions with relevant proteins were performed to understand the behavior and mode of action of this complex in the biological environment. Electrochemical and fluorescence studies showed that TM85 strongly binds to albumin. Studies carried out to study the formation of TM85 which adducts with ubiquitin and cytochrome c were performed by electrospray ionization mass spectrometry (ESI-MS). Antitumor activity was evaluated against a variety of human cancer cell lines, namely A2780, A2780cisR, MCF7, MDAMB231, HT29, PC3 and V79 non-tumorigenic cells and compared with the reference drug cisplatin. TM85 cytotoxic effect was reduced in the presence of endocytosis modulators at low temperatures, suggesting an energy-dependent mechanism consistent with endocytosis. Ultrastructural analysis by transmission electron microscopy (TEM) revealed that TM85 targets the endomembranar system disrupting the Golgi and also affects the mitochondria. Disruption of plasma membrane observed by flow cytometry could lead to cellular damage and cell death. On the whole, the biological activity evaluated herein combined with the water solubility property suggests that complex TM85 could be a promising anticancer agent. (C) 2013 Elsevier Inc. All rights reserved.