Enzymatic Spermine Metabolites Induce Apoptosis Associated with Increase of p53, caspase-3 and miR-34a in Both Neuroblastoma Cells, SJNKP and the N-Myc-Amplified Form IMR5.

Neuroblastoma (NB) is a common malignant solid tumor in children and accounts for 15% of childhood cancer mortality. Amplification of the N-Myc oncogene is a well-established poor prognostic marker in NB patients and strongly correlates with higher tumor aggression and resistance to treatment. New therapies for patients with N-Myc-amplified NB need to be developed. After treating NB cells with BSAO/SPM, the detection of apoptosis was determined after annexin V-FITC labeling and DNA staining with propidium iodide. The mitochondrial membrane potential activity was checked, labeling cells with the probe JC-1 dye. We analyzed, by real-time RT-PCR, the transcript of genes involved in the apoptotic process, to determine possible down- or upregulation of mRNAs after the treatment on SJNKP and the N-Myc-amplified IMR5 cell lines with BSAO/SPM. The experiments were carried out considering the proapoptotic genes Tp53 and caspase-3. After treatment with BSAO/SPM, both cell lines displayed increased mRNA levels for all these proapoptotic genes. Western blotting analysis with PARP and caspase-3 antibody support that BSAO/SPM treatment induces high levels of apoptosis in cells. The major conclusion is that BSAO/SPM treatment leads to antiproliferative and cytotoxic activity of both NB cell lines, associated with activation of apoptosis.

Aged garlic extract and its constituent, S-allyl-L-cysteine, induce the apoptosis of neuroblastoma cancer cells due to mitochondrial membrane depolarization.

Aged garlic extract (AGE) has been demonstrated to have therapeutic properties in tumors; however its mechanisms of action have not yet been fully elucidated. A previous study revealed that AGE exerts an anti-proliferative effect on a panel of both sensitive [wild-type (WT)] and multidrug-resistant (MDR) human cancer cells. Following treatment of the cells with AGE, cytofluorimetric analysis revealed the occurrence of dose-dependent mitochondrial membrane depolarization (MMD). In this study, in order to further clarify the mechanisms of action of AGE, the effects of AGE on mitochondria isolated from rat liver mitochondria (RLM) were also examined. AGE induced an effect on the components of the electrochemical gradient (ΔµH +), mitochondrial membrane potential (ΔΨm) and mitochondrial electrochemical gradient (ΔpHm). The mitochondrial membrane dysfunctions of RLM induced by AGE, namely the decrease in both membrane potential and chemical gradient were associated with a higher oxidation of both the endogenous glutathione and pyridine nucleotide content. To confirm the anti-proliferative effects of AGE, experiments were performed on the human neuroblastoma (NB) cancer cells, SJ-N-KP and the MYCN-amplified IMR5 cells, using its derivative S-allyl-L-cysteine (SAC), with the aim of providing evidence of the anticancer activity of this compound and its possible molecular mechanism as regards the induction of cytotoxicity. Following treatment of the cells with SAC at 20 mM, cell viability was determined by MTT assay and apoptosis was detected by flow cytometry, using Annexin V-FITC labeling. The percentages of cells undergoing apoptosis was found to be 48.0% in the SJ-N-KP and 50.1% in the IMR5 cells. By cytofluorimetric analysis, it was suggested that the target of SAC are the mitochondria. Mitochondrial activity was examined by labeling the cells with the probe, 5,5',6,6'-tetrachloro-1,1',3,3'-tetraethylimidacarbocyanine iodide (JC-1). Following treatment with SAC at 50 mM, both NB cell lines exhibited a marked increase in MMD. On the whole, the findings of this study indicate that both natural products, AGE and SAC, cause cytotoxicity to tumor cells via the induction of mitochondrial permeability transition (MPT).

Involvement of the biogenic active amine agmatine in mitochondrial membrane permeabilization and release of pro-apoptotic factors.

Agmatine (AGM) produces a dual effect on the mitochondrial permeability transition (MPT) mechanism in rat liver mitochondria: at low concentrations, it induces the phenomenon, at high ones, inhibits it. The prevention at high concentrations is evidenced by the significant inhibition of mitochondrial swelling induced by Ca2+ and phosphate; in this condition, AGM both prevents the release of Apoptosis Inducing Factor (AIF) and enhances the release of other pro-apoptotic factors, such as cytochrome c (cyt c) and Smac/DIABLO. As these factors are released without MPT induction, the involvement of mitochondrial outer membrane permeabilization (MOMP) could be hypothesized. Cyclosporin A (CsA), a powerful inhibitor of MPT, enhanced the AGM-mediated inhibition of swelling, and surprisingly, prevented the release of cyt c and Smac/DIABLO. In the presence of Ca2+, AGM also activated the Bcl-2 family protein Bax, a key factor in inducing MOMP, which is inactivated by CsA. Together with the voltage-dependent anion channel (VDAC), Bax forms channels in the outer membrane further supporting the involvement of MOMP in the release of pro-apoptotic factors. In view of the fact that VDAC was inactivated by ruthenium red, which in turn inhibited the release of cyt c, it can be hypothesized that, on the one hand, AGM inhibits MPT induction and, on the other, it selectively permeabilizes the outer membrane via MOMP induction.

Link between spermine oxidase and apoptosis antagonizing transcription factor: A new pathway in neuroblastoma.

Neuroblastoma (NB) is a heterogeneous extra­cranial childhood type of cancer, responsible for approximately 15% of all paediatric cancer­related deaths. Although several critical genetic aberrations have been related to NB, only a few established molecular markers have been associated with prognosis [V­myc avian myelocytomatosis viral oncogene (MYCN) locus amplification, deletions of part of chromosome 1p, 11q23 and gain of 17q]. Regrettably, direct evidence of NB­related tumour suppressors or oncogenes has not been currently identified at these chromosomal regions. MYCN locus amplification is present in approximately 20­30% of cases and is associated with a poor clinical outcome, representing the most important genetic prognostic marker. The functional guidelines for the prognosis of NB identify high­risk patients (<40% survival probabilities), but fail to identify patients at low and intermediate stages of the disease, which remains an issue to be resolved in NB. It has been shown that in NB cell lines and in a total­spermine oxidase (SMOX) transgenic mouse model, SMOX overexpression induces cellular stress via reactive oxygen species (ROS) imbalance. In this study, we demonstrated that the high expression level of the cytoprotective gene, apoptosis-antagonizing transcription factor (AATF), was driven by SMOX gene overexpression in both NB cells and Total­SMOX mice. The anti­apoptotic effect of AATF was supported by analysing the inhibition of the expression of the pro­apoptotic genes, BAX, BAK and PUMA, which were decreased, in both the in vitro and in vivo SMOX overexpressing model systems investigated. On the whole, this study supports the hypothesis that the SMOX gene can be considered as a novel anti­apoptotic marker in NB.

Polyamine Metabolism as a Therapeutic Target inHedgehog-Driven Basal Cell Carcinomaand Medulloblastoma.

Hedgehog (Hh) signaling is a critical developmental regulator and its aberrant activation,due to somatic or germline mutations of genes encoding pathway components, causes Basal CellCarcinoma (BCC) and medulloblastoma (MB). A growing effort has been devoted at theidentification of druggable vulnerabilities of the Hedgehog signaling, leading to the identificationof various compounds with variable efficacy and/or safety. Emerging evidence shows that anaberrant polyamine metabolism is a hallmark of Hh-dependent tumors and that itspharmacological inhibition elicits relevant therapeutic effects in clinical or preclinical models ofBCC and MB. We discuss here the current knowledge of polyamine metabolism, its role in cancerand the available targeting strategies. We review the literature about the connection betweenpolyamines and the Hedgehog signaling, and the potential therapeutic benefit of targetingpolyamine metabolism in two malignancies where Hh pathways play a well-established role: BCCand MB.

Endogenous and food-derived polyamines: determination by electrochemical sensing.

Polyamines (PAs) are involved in a variety of fundamental physio-pathologic processes. The concentration of these polycations in organs and tissues depends on their endogenous production and oxidation rates, and on their intake from foods. Besides being largely accepted as markers for the progress of several pathologies, PAs may exert themselves different effects on humans, ranging from being positive to be drastically detrimental depending on the organism conditions. Thus, if the determination of polyamines content in tissue samples is of great importance as they could be indicators of several diseases, their quantification in food is fundamental for modulating the diet to respond to a specific human health status. Thus, the determination of PA content in food is increasingly urgent. Standard analytical methods for polyamine quantification are mainly based on chromatography, where high-performance liquid chromatography and gas chromatography are the most often used, involving pre-column or post-column derivatization techniques. Driven by the growing need for rapid in situ analyses, electrochemical biosensors, comprising various combinations of different enzymes or nanomaterials for the selective bio-recognition and detection, are emerging as competitors of standard detection systems. The present review is aimed at providing an up-to-date overview on the recent progresses in the development of sensors and biosensors for the detection of polyamines in human tissues and food samples. Basic principles of different electrochemical (bio)sensor formats are reported and the applications in human tissues and in foods was evidenced.

The antioxidant, aged garlic extract, exerts cytotoxic effects on wild-type and multidrug-resistant human cancer cells by altering mitochondrial permeability.

Aged garlic extract (AGE) has been shown to possess therapeutic properties in cancer; however its mechanisms of action are unclear. In this study, we demonstrate by MTT assay that AGE exerts an anti-proliferative effect on a panel of both sensitive and multidrug-resistant (MDR) human cancer cell lines and enhances the effects of hyperthermia (42˚C) on M14 melanoma cells. The evaluation of the mitochondrial activity in whole cancer cells treated with AGE, performed by cytofluorimetric analysis in the presence of the lipophilic cationic fluorochrome JC-1, revealed the occurrence of dose-dependent mitochondrial membrane depolarization. Membrane potential was measured by the TPP+ selective electrode. In order to shed light on its mechanisms of action, the effects of AGE on isolated rat liver mitochondria were also examined. In this regard, AGE induced a mitochondrial membrane hyperpolarization of approximately 15 mV through a mechanism that was similar to that observed with the ionophores, nigericin or salinomycin, by activating an exchange between endogenous K+ with exogenous H+. The prolonged incubation of the mitochondria with AGE induced depolarization and matrix swelling, indicative of mitochondrial permeability transition induction that, however, occurs through a different mechanism from the well-known one. In particular, the transition pore opening induced by AGE was due to the rearrangement of the mitochondrial membranes following the increased activity of the K+/H+ exchanger. On the whole, the findings of this study indicate that AGE exerts cytotoxic effects on cancer cells by altering mitochondrial permeability. In particular, AGE in the mitochondria activates K+/H+ exchanger, causes oxidative stress and induces mitochondrial permeability transition (MPT).

Spectroscopic and calorimetric characterization of spermine oxidase and its association forms.

Spermine oxidase (SMOX) is a flavin-containing enzyme that oxidizes spermine to produce spermidine, 3-aminopropanaldehyde, and hydrogen peroxide. SMOX has been shown to play key roles in inflammation and carcinogenesis; indeed, it is differentially expressed in several human cancer types. Our previous investigation has revealed that SMOX purified after heterologous expression in Escherichia coli actually consists of monomers, covalent homodimers, and other higher-order forms. All association forms oxidize spermine and, after treatment with dithiothreitol, revert to SMOX monomer. Here, we report a detailed investigation on the thermal denaturation of SMOX and its association forms in native and reducing conditions. By combining spectroscopic methods (circular dichroism, fluorescence) and thermal methods (differential scanning calorimetry), we provide new insights into the structure, the transformation, and the stability of SMOX. While the crystal structure of this protein is not available yet, experimental results are interpreted also on the basis of a novel SMOX structural model, obtained in silico exploiting the recently solved acetylspermine oxidase crystal structure. We conclude that while at least one specific intermolecular disulfide bond links two SMOX molecules to form the homodimer, the thermal denaturation profiles can be justified by the presence of at least one intramolecular disulfide bond, which also plays a critical role in the stabilization of the overall three-dimensional SMOX structure, and in particular of its flavin adenine dinucleotide-containing active site.

Aged garlic extract exerts endothelium-dependent vasorelaxant effect on rat aorta by increasing nitric oxide production.

BACKGROUND: Clinical trials have shown that aged garlic extract (AGE) is effective in reducing blood pressure of hypertensive patients. However, the mechanisms involved remain to be elucidated. PURPOSE: The aim of the present study was to investigate the vasorelaxant effect of AGE on the aorta and its mechanism of action in order to clarify the blood pressure-lowering action of AGE. METHODS: The vasorelaxant effect was evaluated in isolated rat aortic rings. After aortic rings were contracted by 3 × 10-6M norepinephrine (NE) for 30min, AGE and other test drugs were added to the aortic rings. All results were expressed as percentages of the maximal NE-induced contraction. RESULTS: AGE induced the concentration-dependent vasorelaxation of isolated rat aortic rings that had been precontracted with norepinephrine. The effect of AGE was severely impaired in aortic rings lacking endothelium. In addition, the effect of AGE was inhibited by a nitric oxide synthase (NOS) inhibitor and a nitric oxide (NO) scavenger. Moreover, AGE treatment of aorta significantly increased the NO production. When various constituents of AGE were tested, the vasorelaxation of aorta was observed only in the presence of L-arginine, a substrate of NOS. CONCLUSION: AGE causes endothelium-dependent vasorelaxation of aorta via stimulation of NO production and that L-arginine in AGE serves as a key agent for NOS-mediated NO production.

Dehydrodiconiferyl alcohol suppresses monocyte adhesion to endothelial cells by attenuation of JNK signaling pathway.

Several clinical studies have shown that the intake of aged garlic extract improves endothelial dysfunction. Lignan compounds, (+)-(2S,3R)-dehydrodiconiferyl alcohol (DDC) and (-)-(2R,3S)-dihydrodehydrodiconiferyl alcohol (DDDC), have been isolated as antioxidants in aged garlic extract. There is evidence showing the importance of oxidative stress in endothelial dysfunction. In the present study, we examined whether DDC and DDDC enhance endothelial cell function in vitro. Cell adhesion assay was performed using THP-1 monocyte and human umbilical vein endothelial cells (HUVECs) which were activated by lipopolysaccharide (LPS) or advanced glycation end products (AGEs)-BSA. Cellular ELISA method was used for the evaluation of vascular cell adhesion molecule 1 (VCAM-1) expression on HUVECs. DDC and DDDC suppressed the adhesion of THP-1 to HUVECs which was activated by LPS or AGEs-BSA. DDC and DDDC also inhibited VCAM-1 expression induced by LPS or AGEs-BSA, but DDDC was less effective than DDC. In addition, the inhibitory effect of DDC on VCAM-1 expression involved suppressing JNK/c-Jun pathway rather than NF-κB pathway. DDC has an inhibitory effect on VCAM-1 expression via JNK pathway in endothelial cells and therefore may serve as a novel pharmacological agent to improve endothelial dysfunction.