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Studies of cytotoxicity on human tumor cells induced by enzymatic oxidation products of polyamines: a new approach in cancer therapy
No full textBackground
The polyamines spermine, spermidine and putrescine are ubiquitous cell components and required for several physiological functions, such as DNA synthesis, cell growth and differentiation. Polyamine concentrations are high in growing tissues such as tumors. Amine oxidases are important because they contribute to the regulation of polyamines’ levels. These enzymes catalyze the oxidative deamination of polyamines to generate the reaction products H2O2 and aldehyde(s), that are able to induce oxidative stress in several cultured human tumor cell lines. Because of the limited success of chemotherapy for tumors, most investigations have oriented on cellular and molecular mechanisms responsible for the onset of multidrug resistance (MDR). Several studies have therefore been performed to overcome the MDR phenotype and to develop innovative chemotherapeutic strategies effective against MDR tumors. Our previous studies using bovine serum amine oxidase (BSAO) have shown that H2O2 and aldehyde(s) were responsible for the cytotoxicity induced in human colon adenocarcinoma cells (LoVo). The treatment of tumors with BSAO could be useful as a new strategy for cancer therapy. In this study, maize polyamine oxidase (ZmPAO), a flavin adenine dinucleotide (FAD)-dependent enzyme, was used due to both its high enzymatic specific activity and the low molecular weight, which are important aspects for the delivery of these enzymes into cancer cells. I investigated on the cytotoxicity and the mechanism of cell death induced by enzymatic oxidation product of polyamine generated by ZmPAO.
Methods
Subconfluent LoVo WT cells, its doxorubicin resistance (MDR) cells, and AGS gastric tumor cells were used to investigate the cytotoxic effect induced by polyamine metabolites. The harvested tumor cells were treated with exogenous spermine and ZmPAO purified from maize shoot at 37°C up to 1 hr. The treated cells were used in the following assays. Cell viability assays were performed by clonogenic assay. After the treatment, cells were incubated at 37°C for 10-17 days until colonies were formed. The colonies were stained and counted. The involvement of apoptotic cell death was assessed by Annexin V-FITC/PI staining and cell cycle, using flow cytometry. An increasing of the sub-G1 peak was detected. In order to determine the involvement of mitochondria in apoptosis, we also examined the loss of mitochondrial membrane potential using JC-1. Scanning electron microscopy (SEM) and transmission electron microscopy (TEM) observations were performed on LoVo cells to examine the morphological changes and the ultrastructure modifications, respectively, after the treatment.
Results and discussion
Spermine and ZmPAO treatment reduced the number of colonies in a spermine dose-dependent manner and a time-dependent manner in LoVo and AGS cells. I also found that the treatment further decreased the cell viability in LoVo MDR cells when compared with LoVo WT cells. Moreover, there was no difference in cell viability after the treatment with spermine and spermidine in both LoVo WT and MDR cells, suggesting that aminoaldehydes derived from spermine and spermidine do not have any cytotoxic effect, it is mainly due to H2O2. Further colony formation assay revealed that cytotoxicity induced by spermine metabolites was completely prevented by the addition of exogenous catalase in both LoVo WT and MDR cells, while it was not affected by the presence of exogenous ALDH. Moreover, LoVo cells exposed to exogenous H2O2 exhibited a slightly higher cytotoxicity than that induced by ZmPAO and spermine. The concentration of H2O2 actually generated by ZmPAO from spermine might be lower than that of H2O2 when directly exposed. Similar results were also obtained in AGS cells, showing that the effect of cell growth inhibition induced by polyamines and ZmPAO was not cell specific.
Treatment with ZmPAO and spermine at 42°C showed an increase in cytotoxicity on LoVo and AGS cells when compared with the cells treated at 37°C. Cytotoxicity was higher in LoVo MDR cells than in their WT counterpart, and it is probably due to a synergistic effect of spermine metabolites at 42°C.
Annexin V-FITC and PI staining assay showed that the treatment with ZmPAO and spermine increased the percentage of apoptotic cells population in a spermine dose-dependent manner in LoVo WT. The treatment also increased the cell population at the sub-G1 phase that is characteristic of apoptosis. In both assays, the proportion of apoptotic cells was higher in LoVo MDR than in LoVo WT.
SEM analysis showed a significant number of membrane blebs after the treatment with ZmPAO and spermine in LoVo MDR cells, while LoVo WT cells showed minor morphological changes. TEM observation further revealed that the treatment exhibited the typical ultrastructural features of apoptosis, including vacuolization in the cytoplasm and mitochondrial alterations. In particular, in MDR cells, I found severe vacuolization and cell membrane blebbing.
The involvement of mitochondria in the cytotoxicity was supported by JC-1 assay. It showed that the exposure of LoVo cells to ZmPAO and spermine reduced mitochondrial membrane potential, which was greater in LoVo MDR cells than in WT cells, in agreement with the results of cell viability and apoptosis assays.
We demonstrated that ZmPAO and spermine treatment induced cytotoxicity through an apoptotic pathway, which was more pronounced in LoVo MDR cells compared to LoVo WT ones. Since polyamine levels are up-regulated in tumor sites and the development of MDR tumor cells is recognized as a major problem, the design of a new therapeutic strategy based on the use of this combination in human tumors might be taken into account, making this approach mainly attractive in treating MDR cancer patients
Milestones and recent discoveries on cell death mediated by mitochondria and their interactions with biologically active amines
No full textMitochondria represent cell “powerhouses,” being involved in energy transduction from the electrochemical gradient to ATP synthesis. The morphology of their cell types may change, according to various metabolic processes or osmotic pressure. A new morphology of the inner membrane and mitochondrial cristae, significantly different from the previous one, has been proposed for the inner membrane and mitochondrial cristae, based on the technique of electron tomography. Mitochondrial Ca2+ transport (the transporter has been isolated) generates reactive oxygen species and induces the mitochondrial permeability transition of both inner and outer mitochondrial
membranes, leading to induction of necrosis and apoptosis. In the mitochondria of several cell types (liver, kidney, and heart), mitochondrial oxidative stress is an essential step in the induction of cell death, although not in brain, in which the phenomenon is caused by a different mechanism
Pathophysiological implications of mitochondrial oxidative stress mediated by mitochondriotropic agents and polyamines: The role of tyrosine phosphorylation
No full textMitochondria, once merely considered as the "powerhouse" of cells, as they generate more than 90 % of cellular ATP, are now known to play a central role in many metabolic processes, including oxidative stress and apoptosis. More than 40 known human diseases are the result of excessive production of reactive oxygen species (ROS), bioenergetic collapse and dysregulated apoptosis. Mitochondria are the main source of ROS in cells, due to the activity of the respiratory chain. In normal physiological conditions, ROS generation is limited by the anti-oxidant enzymatic systems in mitochondria. However, disregulation of the activity of these enzymes or interaction of respiratory complexes with mitochondriotropic agents may lead to a rise in ROS concentrations, resulting in oxidative stress, mitochondrial permeability transition (MPT) induction and triggering of the apoptotic pathway. ROS concentration is also increased by the activity of amine oxidases located inside and outside mitochondria, with oxidation of biogenic amines and polyamines. However, it should also be recalled that, depending on its concentration, the polyamine spermine can also protect against stress caused by ROS scavenging. In higher organisms, cell signaling pathways are the main regulators in energy production, since they act at the level of mitochondrial oxidative phosphorylation and participate in the induction of the MPT. Thus, respiratory complexes, ATP synthase and transition pore components are the targets of tyrosine kinases and phosphatases. Increased ROS may also regulate the tyrosine phosphorylation of target proteins by activating Src kinases or phosphatases, preventing or inducing a number of pathological states
Ser/Thr kinases and polyamines in the regulation of non-canonical functions of elongation factor 1A
No full textThe link between eukaryotic translation elongation factor 1A (eEF1A) and signal transduction pathways through the regulatory mechanism of phosphorylation has never been considered. In this review, we focus on the different kinases that recognize the Ser and Thr residues of the eEF1A1 and eEF1A2 isoforms and regulate their involvement in different cellular processes like cell survival and apoptosis. In this context, polyamines seem to play a role in the regulation of the translation elongation process by modulating the Ser/Thr kinases involved in the phosphorylation of translation elongation factors
Cytotoxic effect induced by maize polyamine oxidase in presence of spermine in human cancer cells.
No full text
Going Beyond Counting First Authors in Author Co-citation Analysis
Get PDFThe present study examines one of the fundamental aspects of author co-citation analysis (ACA) - the way co-citation
counts are defined. Co-citation counting provides the data on which all subsequent statistical analyses and mappings
are based, and we compare ACA results based on two different types of co-citation counting - the traditional type that
only counts the first one among a cited work's authors on the one hand and a non-traditional type that takes into
account the first 5 authors of a cited work on the other hand. Results indicate that the picture produced through this non-traditional author co-citation counting contains more coherent author groups and is therefore considerably clearer. However, this picture represents fewer specialties in the research field being studied than that produced through the traditional first-author co-citation counting when the same number of top-ranked authors is selected and analyzed. Reasons for these effects are discussed
Variations on the Author
Get PDF“Variations on the Author” discusses two of Eduardo Coutinho’s recent films (Um Dia na Vida, from 2010, and Últimas Conversas, posthumously released in 2015) and their contribution to the general question of documentary authorship. The director’s filmography is characterized by a consistent yet self-effacing form of authorial self-inscription: Coutinho often features as an interviewer that rather than express opinions propels discourses; an interviewer that is good at listening. This mode of self-inscription characterizes him as an author who is not expressive but who is nonetheless markedly present on the screen. In Um Dia na Vida, however, Coutinho is completely absent form the image, while Últimas Conversas, on the contrary, includes a confessional prologue that moves the director from the margins to the center of his films. This article examines the ways in which these works stand out in the filmography of a director who offers new insights into the notion of cinematic authorship
Appropriate Similarity Measures for Author Cocitation Analysis
Get PDFWe provide a number of new insights into the methodological discussion about author cocitation analysis. We first argue that the use of the Pearson correlation for measuring the similarity between authors’ cocitation profiles is not very satisfactory. We then discuss what kind of similarity measures may be used as an alternative to the Pearson correlation. We consider three similarity measures in particular. One is the well-known cosine. The other two similarity measures have not been used before in the bibliometric literature. Finally, we show by means of an example that our findings have a high practical relevance.information science;Pearson correlation;cosine;similarity measure;author cocitation analysis
Stability of spermine oxidase to thermal and chemical denaturation: comparison with bovine serum amine oxidase
No full textSpermine oxidase (SMOX) is a flavin-containing enzyme that specifically oxidizes spermine to produce spermidine, 3-aminopropanaldehyde and hydrogen peroxide. While no crystal structure is available for any mammalian SMOX, X-ray crystallography showed that the yeast Fms1 polyamine oxidase has a dimeric structure. Based on this scenario, we have investigated the quaternary structure of the SMOX protein by native gel electrophoresis, which revealed a composite gel band pattern, suggesting the formation of protein complexes. All high-order protein complexes are sensitive to reducing conditions, showing that disulfide bonds were responsible for protein complexes formation. The major gel band other than the SMOX monomer is the covalent SMOX homodimer, which was disassembled by increasing the reducing conditions, while being resistant to other denaturing conditions. Homodimeric and monomeric SMOXs are catalytically active, as revealed after gel staining for enzymatic activity. An engineered SMOX mutant deprived of all but two cysteine residues was prepared and characterized experimentally, resulting in a monomeric species. High-sensitivity differential scanning calorimetry of SMOX was compared with that of bovine serum amine oxidase, to analyse their thermal stability. Furthermore, enzymatic activity assays and fluorescence spectroscopy were used to gain insight into the unfolding process
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