Lena Ruiz-Azuara

Copper Compounds in Cancer Chemotherapy

Transitional metals have a large variety of coordination numbers and geometries, accessible redox states in physiological conditions and a wide range of thermodynamic and reactivity properties which can be successfully tuned by selection of suitable ligands. These characteristics can be used to develop new drugs with numerous advantages over the organic based drugs. Historically, research in this field has focus on platinum and DNA targeting; however, anticancer drug research may be expanded to include alternative metal compounds with different mode of action resulting in markedly different cytotoxic response profiles. Cooper complexes with selected ligands are being extensively studied as agents for the treatment of cancer. Current research on copper compounds as antitumoral compounds is being reviewed in this chapter particularly focused on the family of copper Casiopeinas.

Genotoxicity of the copper antineoplastic coordination complexes casiopeinas.

Casiopeinas is the generic name of a group of coordination complexes with a central copper atom bound to organic ligands, designed to be an alternative to cancer therapy. Indeed, some of these compounds can reduce implanted tumors in mice. Casiopeinas were expressly designed to interact with the genetic material, so the aim of the present work is to determine if these compounds have genotoxic activity. The results indicate that casiopeinas produce DNA fragmentation and base oxidation and suggest that their mode of action is related to reactive oxygen species (ROS) generation after copper reduction.

Copper compound induces autophagy and apoptosis of glioma cells by reactive oxygen species and jnk activation

BackgroundGlioblastoma multiforme (GBM) is the most aggressive of the primary brain tumors, with a grim prognosis despite intensive treatment. In the past decades, progress in research has not significantly increased overall survival rate.MethodsThe in vitro antineoplastic effect and mechanism of action of Casiopeina III-ia (Cas III-ia), a copper compound, on rat malignant glioma C6 cells was investigated.ResultsCas III-ia significantly inhibited cell proliferation, inducing autophagy and apoptosis, which correlated with the formation of autophagic vacuoles, overexpression of LC3, Beclin 1, Atg 7, Bax and Bid proteins. A decrease was detected in the mitochondrial membrane potential and in the activity of caspase 3 and 8, together with the generation of intracellular reactive oxygen species (ROS) and increased activity of c-jun NH2-terminal kinase (JNK). The presence of 3-methyladenine (as selective autophagy inhibitor) increased the antineoplastic effect of Cas III-ia, while Z-VAD-FMK only showed partial protection from the antineoplastic effect induced by Cas III-ia, and ROS antioxidants (N-acetylcysteine) decreased apoptosis, autophagy and JNK activity. Moreover, the JNK –specific inhibitor SP600125 prevented Cas III-ia-induced cell death.ConclusionsOur data suggest that Cas III-ia induces cell death by autophagy and apoptosis, in part due to the activation of ROS –dependent JNK signaling. These findings support further studies of Cas III-ia as candidate for treatment of human malignant glioma.

Intercalation processes of copper complexes in DNA

The family of anticancer complexes that include the transition metal copper known as Casiopeínas® shows promising results. Two of these complexes are currently in clinical trials. The interaction of these compounds with DNA has been observed experimentally and several hypotheses regarding the mechanism of action have been developed, and these include the generation of reactive oxygen species, phosphate hydrolysis and/or base-pair intercalation. To advance in the understanding on how these ligands interact with DNA, we present a molecular dynamics study of 21 Casiopeínas with a DNA dodecamer using 10 μs of simulation time for each compound. All the complexes were manually inserted into the minor groove as the starting point of the simulations. The binding energy of each complex and the observed representative type of interaction between the ligand and the DNA is reported. With this extended sampling time, we found that four of the compounds spontaneously flipped open a base pair and moved inside the resulting cavity and four compounds formed stacking interactions with the terminal base pairs. The complexes that formed the intercalation pocket led to more stable interactions.

Casiopeinas IIgly and IIIia Induce Apoptosis in Medulloblastoma Cells

The medulloblastoma is a tumor of the central nervous system that is expressed in childhood. Casiopeinas® are a family of molecules with an active copper 2+ core and an amino acid sequence that seem give them tumoral specificity. The mechanism of action is poorly understood; however, it has been reported that some metals such as copper and some of their complexes are toxic due to their high potential to participate in redox reactions which could cause apoptosis in medulloblastoma cells. Cell survival was measured by the MTT method and apoptosis was identified by the presence of condensed nuclei, disruption of the mitochondrial transmembrane potential, and cytoskeleton disorder. In all cases medulloblastoma cells treated with Casiopeinas showed more apoptotic features than untreated cells. Casiopeinas IIgly and IIIia promise to be important compounds for the treatment of medulloblastoma, mainly by their ability to induce apoptosis.

Casiopeína IIgly induced cytotoxicity to HeLa cells depletes the levels of reduced glutathione and is prevented by dimethyl sulfoxide

The newly synthesized copper coordination compound Casiopeína IIgly (Cas IIgly) is a promising alternative drug in the treatment of cancer, since it has shown cytotoxicity and genotoxicity in different tumour models. Given its enhanced effects after ascorbic acid-mediated copper reduction, Cas IIglys activity is thought to be related to oxidative damage. In the present work, oxidized Cas IIgly failed to induce cytosolic oxidative damage in HeLa cells (only 0.9% of the cell population), and in 2.3% of the treated cells when previously reduced, as evaluated through the oxidation of dihydrorhodamine 123 (DHR 123). However, it showed cytotoxicity, since HeLa cells treated with 10-80 microg/mL Cas IIgly proliferated only at 30% of their normal rate, and at 15% when treated with reduced Cas IIgly. This cytotoxicity is strongly abolished in the presence of the hydroxyl scavenger dimethyl sulfoxide. The decrease, from 3994 to 530 nanograms of reduced glutathione (GSH) per million cells after treatment with 80 microg/mL Casiopeína IIgly, indicates that this drug causes the expenditure of this naturally occurring antioxidant. These results altogether suggest that, albeit Cas IIgly induced cytotoxicity is not related to cytosolic DHR 123 oxidation, it may be related to oxidative damage.

Comparison between the antiproliferative effect and intracellular glutathione depletion induced by Casiopeína IIgly and cisplatin in murine melanoma B16 cells.

BACKGROUND Casiopeína IIgly (Cas IIgly) [Cu(4,7-dimethyl-1,10-phenanthroline)(glycinate)]NO(3) induce oxidative damage in different human tumour cell strains, as the known anticancer agent cisplatin (CDDP) does. PURPOSE To compare glutathione (GSH) depletion induced by Cas IIgly and CDDP in murine melanoma B16 cells and its relationship with their antiproliferative effect. MATERIALS AND METHODS Cell growth was determined according to the sulforhodamine B assay. Intracellular GSH levels were measured by the reduction of Ellmans reagent (DTNB). RESULTS Cas IIgly IC50 in B16 cells was 54.5 μM (24.21 μg/mL), which depleted GSH from 1092 to 585 ng per million cells in a 30 min incubation period. In the other hand, CDDP was less toxic at the same conditions with an IC50 equal to 197.76 μM (59.33 μg/mL), and depleted GSH to 50% of the normal only after a longer exposure period (4h). The addition of 1.8mM ascorbic acid (Asc) or 1mM buthionine sulfoximine (BSO) enhanced Cas IIgly toxicity, whereas it was prevented by 100 U/mL catalase. BSO sensitised B16 cells to CDDP, but neither Asc or catalase modified CDDP effects. CONCLUSIONS The antiproliferative effect of both drugs correlated to intracellular GSH levels. Unlike CDDP, GSH depletion induced by Cas IIgly occurs earlier, it is enhanced by ascorbic acid and preventable by catalase. Redox cycles, feasible only with Cas IIgly, may be an important difference in their mode of action.

Whole Genome Gene Expression Analysis Reveals Casiopeína-Induced Apoptosis Pathways

Copper-based chemotherapeutic compounds Casiopeínas, have been presented as able to promote selective programmed cell death in cancer cells, thus being proper candidates for targeted cancer therapy. DNA fragmentation and apoptosis–in a process mediated by reactive oxygen species–for a number of tumor cells, have been argued to be the main mechanisms. However, a detailed functional mechanism (a model) is still to be defined and interrogated for a wide variety of cellular conditions before establishing settings and parameters needed for their wide clinical application. In order to shorten the gap in this respect, we present a model proposal centered in the role played by intrinsic (or mitochondrial) apoptosis triggered by oxidative stress caused by the chemotherapeutic agent. This model has been inferred based on genome wide expression profiling in cervix cancer (HeLa) cells, as well as statistical and computational tests, validated via functional experiments (both in the same HeLa cells and also in a Neuroblastoma model, the CHP-212 cell line) and assessed by means of data mining studies.

Pharmacokinetics and hematotoxicity of a novel copper-based anticancer agent: casiopeina III-Ea, after a single intravenous dose in rats.

Casiopeina III‐Ea is a mixed chelate copper (II) complex that has shown cytotoxic and antitumor activity in vitro and in vivo. The aim of this study was to investigate the pharmacokinetics of total copper derived from casiopeina III‐Ea administered by intravenous bolus injection to Wistar rats. Other objective was to evaluate the hematotoxicity produced by this compound in those animals. Wistar rats received a single intravenous dose of 4 mg/kg of casiopeina III‐Ea. Blood samples were taken and pharmacokinetics evaluated. Furthermore, erythrocyte copper levels were determined to identify a potential target and Zn levels were analyzed to determine a possible change. For the evaluation of hematotoxicity, both blood and urine samples were collected for hematological and biochemical analyses; moreover, Fe determination was performed. Blood copper and zinc levels, red blood cell copper levels as well as copper, zinc, and iron amounts excreted into urine were analyzed by ICP‐MS. The blood concentration–time profile of copper derived from casiopeina III‐Ea was fitted to a two‐compartment model with a zero‐order input. Cumulative amounts of Cu, Zn, and Fe excreted into rat urine after administration of casiopeina III‐Ea were different with respect to control. Hematological and biochemical data indicated a hemolytic toxicity. Pharmacokinetic analysis of total copper derived from casiopeina III‐Ea provided a general knowledge about distribution and elimination process of this compound. Additionally, the systemic exposure of the copper derived from casiopeina III‐Ea accounts for the hematotoxicity of this complex at test dose.

Genotoxic assessment of the copper chelated compounds Casiopeinas: Clues about their mechanisms of action

Casiopeinas is the generic name of a group of copper chelated complexes designed to be used as antineoplastic. Some of these compounds have shown promising results, and in fact, one of them named Casiopeina III-ia has completed preclinical trials and is ready to start clinical phase I in Mexico. As part of the tests that have to be done to every molecule intended to be used in humans, bacterial assays are required because of their sensitivity, speed and reproducibility and among them, Ames test and the SOS Chromotest are widely used to evaluate DNA damage. With the aim to contribute to complete safety information related to genotoxicity and support the hypothesis about their mode of action, four different Casiopeinas (Cas II-gly, Cas III-Ea, Cas III-ia and Cas III-Ha) were tested for genotoxicity with these assays, as well as differential cytotoxicity upon Escherichia coli mutants defectives in some DNA repair mechanisms. However, although it is well known that these molecules produce DNA breakage, the results of the Chromotest and Ames test were negative. Despite this is controversial, a possible explanation is that there is a direct interaction between DNA and the Casiopeinas tested.