Flávia de Castro Pereira

Atypical fluoroquinolone gold(III) chelates as potential anticancer agents: Relevance of DNA and protein interactions for their mechanism of action

Quinolones are known for their antimicrobial and antitumor activities. Gold(III) compounds constitute an emerging class of biologically active substances, of special interest as potential anticancer agents. In this work three gold(III) complexes of the fluoroquinolones antimicrobial agents norfloxacin (NOR), levofloxacin (LEVO) and sparfloxacin (SPAR) were prepared and characterized with physicochemical and spectroscopic techniques. In these complexes, NOR, LEVO and SPAR act as bidentate neutral ligands bound to gold(III) through the nitrogen atoms of the piperazine ring, which is an unusual mode of coordination for this class of compounds. Two chloride ions occupy the remaining coordination sites. The cytotoxic activity of the fluoroquinolones and their gold(III) complexes was tested against the A20 (murine lymphoma), B16-F10 (murine melanoma) and K562 (human myeloid leukemia) tumor cell lines as well as the L919 (murine lung fibroblasts) and MCR-5 (human lung fibroblasts) normal cells lines. All complexes were more active than their corresponding free ligands. Complex [AuCl(2)(LEVO)]Cl was selected for DNA fragmentation and cell cycle analysis. Spectroscopic titration with calf-thymus DNA (CT DNA) showed that the complexes can bind weakly to CT DNA, probably by an external contact (electrostatic or groove binding). The complexes exhibit good binding propensity to bovine serum albumin (BSA) having relatively high binding constant values.

Cytoxicity and Apoptotic Mechanism of Ruthenium(II) Amino Acid Complexes in Sarcoma-180 Tumor Cells

Over the past several decades, much attention has been focused on ruthenium complexes in antitumor therapy. Ruthenium is a transition metal that possesses several advantages for rational antitumor drug design and biological applications. In the present study, five ruthenium complexes containing amino acids were studied in vitro to determine their biological activity against sarcoma-180 tumor cells. The cytotoxicity of the complexes was evaluated by an MTT assay, and their mechanism of action was investigated. The results demonstrated that the five complexes inhibited the growth of the S180 tumor cell line, with IC50 values ranging from 22.53 µM to 50.18 µM, and showed low cytotoxicity against normal L929 fibroblast cells. Flow cytometric analysis revealed that the [Ru(gly)(bipy)(dppb)]PF6 complex (2) inhibited the growth of the tumor cells by inducing apoptosis, as evidenced by an increased number of Annexin V-positive cells and G0/G1 phase cell cycle arrest. Further investigation showed that complex 2 caused a loss of mitochondrial membrane potential; activated caspases 3, caspase-8, and caspase-9 and caused a change in the mRNA expression levels of caspase 3, caspase-9 as well as the bax genes. The levels of the pro-apoptotic Bcl-2 family protein Bak were increased. Thus, we demonstrated that ruthenium amino acid complexes are promising drugs against S180 tumor cells, and we recommend further investigations of their role as chemotherapeutic agents for sarcomas.

Molecular Detection of Adenoviruses in Lakes and Rivers of Goiânia, Goiás, Brazil

Adenoviruses are a highly important public health issue, since they are among the most persistent and ubiquitous viruses present in water and associated with a variety of clinical manifestations. The aim of this study was to use molecular techniques for the detection of adenovirus in public and recreational water supplies in Goiânia, Brazil. From December 2007 to November 2008, 54 water samples were collected in 5 different sites in 2 lakes and 2 rivers of the city. The samples were filtered in a positively charged nylon membrane, and the DNA was extracted using the phenol–chloroform–isoamyl alcohol method. Semi-nested PCR was used to detect adenovirus DNA, and sequence analysis of the semi-nested PCR products was performed to identify the recovered viruses. Adenovirus DNA was detected in 43% (24 of 54) of samples collected. Considering all examined sites, MP1 presented the highest occurrence of adenovirus (6 positive from 10 collected samples), followed by MP2 (3 positive from 6 collected samples), JL (10 positive from 21 collected samples), VB (3 positive from 9 collected samples), and BB (2 positive from 8 collected samples), respectively. The methodology employed proved to be feasible, fast, low-cost, and suitable to be used as screening approach on adenovirus detection in water for public sanitation companies.

The ruthenium complex cis-(dichloro)tetrammineruthenium(III) chloride induces apoptosis and damages DNA in murine sarcoma 180 cells.

Ruthenium(III) complexes are increasingly attracting the interest of researchers due to their promising pharmacological properties. Recently, we reported that the cis-(dichloro)tetrammineruthenium(III) chloride compound has cytotoxic effects on murine sarcoma 180 (S-180) cells. In an effort to understand the mechanism responsible for their cytotoxicity, study we investigated the genotoxicity, cell cycle distribution and induction of apoptosis caused by cis-(dichloro)tetrammineruthenium(III) chloride in S-180 tumour cells. cis-(dichloro)tetrammineruthenium(III) chloride treatment induced significant DNA damage in S-180 cells, as detected by the alkaline comet assay. In the cell cycle analysis, cis-(dichloro)tetrammineruthenium(III) chloride caused an increase in the number of cells in G1 phase, accompanied by a decrease in the S and G2 phases after 24 h of treatment. In contrast, the cell cycle distribution of S-180 cells treated with cis-(dichloro)tetrammineruthenium(III) chloride for 48 h showed a concentration-dependent increase in the sub-G1 phase (indicating apoptosis), with a corresponding decrease in cells in the G1, S and G2 phases. In addition, cis-(dichloro)tetrammineruthenium(III) chloride treatment induced apoptosis in a time-dependent manner, as observed by the increased numbers of annexin V-positive cells. Taken together, these findings strongly demonstrate that DNA damage, cell cycle changes and apoptosis may correlate with the cytotoxic effects of cis-(dichloro)tetrammineruthenium(III) chloride on S-180 cells.

The compound cis-(dichloro)tetrammineruthenium(III) chloride induces caspase-mediated apoptosis in K562 cells

Ruthenium(III) complexes are increasingly attracting the interest of researchers due to their promising pharmacological properties. In the present study, we investigated the ability of cis-(dichloro)tetrammineruthenium(III) chloride to produce lethal effects in human chronic myelogenous leukemia K562 cells. The MTT tetrazolium reduction test and the trypan blue exclusion assay revealed that the IC(50) for the compound after 48 h of incubation with K562 cells was approximately 10.74 and 73.45 microM, respectively. Interestingly, it was observed that this compound exhibits mild cytotoxicity towards MRC-5 human fibroblast cells (IC(50)>383 microM). Flow cytometric analysis revealed that cis-(dichloro)tetrammineruthenium(III) chloride was capable of change cell cycle distribution since the percentage of cells in the G1, S and G2 phases decreased. In addition, treatment with this compound induced apoptotic cell death in K562 cells, demonstrated by increased DNA content in the sub-G1-peak and a significant increase in caspase-3 activity. Assay using cyclosporin A, an inhibitor of the mitochondrial permeability transition pore (MPT) showed that the preincubation of K562 cells with this inhibitor had not effect on cis-(dichloro)tetrammineruthenium(III) chloride induced caspase-3 activation. In summary, cis-(dichloro)tetrammineruthenium(III) chloride displayed a significant cytotoxic effect through cell cycle arrest and apoptotic induction in K562 cells, which suggests that cis-(dichloro)tetrammineruthenium(III) chloride might have therapeutic potential against leukemia.

Cytotoxic and Genotoxic Effects of cis-Tetraammine(oxalato)Ruthenium(III) Dithionate on the Root Meristem Cells of Allium cepa

Ruthenium complexes have attracted much attention as possible building blocks for new transition-metal-based antitumor agents. The present study examines the mitotoxic and clastogenic effects induced in the root tips of Allium cepa by cis-tetraammine(oxalato)ruthenium(III) dithionate {cis-[Ru(C2O2)(NH3)4]2(S2O6)} at different exposure durations and concentrations. Correlation tests were performed to determine the effects of the time of exposure and concentration of ruthenium complex on mitotic index (MI) and mitotic aberration index. A comparison of MI results of cis-[Ru(C2O2)(NH3)4]2(S2O6) to those of lead nitrate reveals that the ruthenium complex demonstrates an average mitotic inhibition eightfold higher than lead, with the frequency of cellular abnormalities almost fourfold lower and mitotic aberration threefold lower. A. cepa root cells exposed to a range of ruthenium complex concentrations did not display significant clastogenic effects. Cis-tetraammine(oxalato)ruthenium(III) dithionate therefore exhibits a remarkable capacity to inhibit mitosis, perhaps by inhibiting DNA synthesis or blocking the cell cycle in the G2 phase. Further investigation of the mechanisms of action of this ruthenium complex will be important to define its clinical potential and to contribute to a novel and rational approach to developing a new metal-based drug with antitumor properties complementary to those exhibited by the drugs already in clinical use.

Cytotoxicity of glass ionomer cements containing silver nanoparticles

Background Some studies have investigated the possibility of incorporating silver nanoparticles (NAg) into dental materials to improve their antibacterial properties. However, the potential toxic effect of this material on pulp cells should be investigated in order to avoid additional damage to the pulp tissue. This study evaluated the cytotoxicity of conventional and resin-modified glass ionomer cements (GIC) with and without addition of NAg. Material and Methods NAg were added to the materials at two different concentrations by weight: 0.1% and 0.2%. Specimens with standardized dimensions were prepared, immersed in 400 µL of culture medium and incubated at 37°C and 5% CO2 for 48 h to prepare GIC liquid extracts, which were then incubated in contact with cells for 48 h. Culture medium and 0.78% NAg solution were used as negative and positive controls, respectively. Cell viability was determined by MTT and Trypan Blue assays. ANOVA and the Tukey test (α=0.05) were used for statistical analyses. Results Both tests revealed a significant decrease in cell viability in all groups of resin modified cements (p<0.001). There were no statistically significant differences between groups with and without NAg (p>0.05). The differences in cell viability between any group of conventional GIC and the negative control were not statistically significant (p>0.05). Conclusions NAg did not affect the cytotoxicity of the GIC under evaluation. Key words:Glass ionomer cements, totoxicity, cell culture techniques, nanotechnology, metal nanoparticles.

Cytotoxic effects of the compound cis-tetraammine(oxalato)ruthenium(III) dithionate on K-562 human chronic myelogenous leukemia cells.

Chemotherapy is a common treatment for leukemia. Ruthenium complexes have shown potential utility in chemotherapy and photodynamic therapy. The identification of new chemotherapeutics agents is critical for further progress in the treatment of leukemia. Ruthenium complexes generally have lower toxicities compared to cisplatin attributed to their specific accumulation in cancer tissues. Based on these evidences, in the present work we studied the cytotoxic activity of the ruthenium(III) compound cis-tetraammine(oxalato)ruthenium(III) dithionate - {cis-[Ru(C2O4)(NH3)4]2(S2O6)} against human chronic myelogenous leukemia cells (K-562) tumor cell line. The tested compound induces cell death in a dose and time dependent manner on K-562 cells. It is found that the effect was improved linearly while prolonging the incubation time. Compared to the cell cycle profiles of untreated cells, flow cytometric analysis indicated the sub-G1 arresting effect of ruthenium compound on K-562 cells. In our study, {cis-[Ru(C2O4)(NH3)4]2(S2O6)} shows a significant increase in tailed cells in any of the concentrations tested compared with negative control. Consequently, the concentration of {cis-[Ru(C2O4)(NH3)4]2(S2O6)} might be associated cytotoxicity with direct effect on K-562 cells DNA. Thus, it can be deducted that ruthenium-based compounds present selectivity to enter both tumor and normal cells. Additional studies are needed to determine the molecular mechanisms of the active components and to evaluate the potential in vivo anticancer activity of the cis-tetraammine(oxalato)ruthenium(III) dithionate.

1H HR-MAS NMR and S180 cells: metabolite assignment and evaluation of pulse sequence

High resolution magic angle spinning 1H nuclear magnetic resonance spectroscopy (HR-MAS NMR) is a useful technique for evaluation of intact cells and tissues. However, optimal NMR parameters are crucial in obtaining reliable results. To identify the key steps for the optimization of HR-MAS NMR parameters, we assessed different pulse sequences and NMR parameters using sarcoma 180 (S180) cells. A complete assignment of the metabolites of S180 is given to assist future studies.

Cytotoxicity and genotoxicity of intravitreal adalimumab administration in rabbit retinal cells

PURPOSE To assess the cytotoxicity and genotoxicity of intravitreal adalimumab treatment in an animal experimental model using cytological and molecular techniques. METHODS Eighteen rabbits were randomly assigned to three groups: control, adalimumab treatment, and placebo. Cytotoxicity on retinal cells was evaluated using flow cytometry assays to determine the level of apoptosis and necrosis. Genotoxicity was evaluated by comet assays to assess DNA damage, and quantitative real-time polymerase chain reaction (qPCR) was used to evaluate expression of apoptosis-inducing caspases (8 and 3). RESULTS No cytotoxicity or genotoxicity was observed in any of the two treatment groups (adalimumab and placebo) following intravitreal administration compared with the control group. Flow cytometry analysis revealed that more than 90% of the cells were viable, and only a low proportion of retinal cells presented apoptotic (~10%) or necrotic (<1%) activity across all groups. Molecular damage was also low with a maximum of 6.4% DNA degradation observed in the comet assays. In addition, no increase in gene expression of apoptosis-inducing caspases was observed on retinal cells by qPCR in both the adalimumab and placebo groups compared with the control group. CONCLUSION The use of adalimumab resulted in no detectable cytotoxicity or genotoxicity on retinal cells for up to 60 days upon administration. These results therefore indicate that adalimumab may be a safe option for intravitreal application to treat ocular inflammatory diseases in which TNF-α is involved.