The role of heat shock proteins in the pathogenesis of heart failure (Review).

The influence of heat shock proteins (HSPs) on protein quality control systems in cardiomyocytes is currently under investigation. The effect of HSPs on the regulated cell death of cardiomyocytes (CMCs) is of great importance, since they play a major role in the implementation of compensatory and adaptive mechanisms in the event of cardiac damage. HSPs mediate a number of mechanisms that activate the apoptotic cascade, playing both pro­ and anti­apoptotic roles depending on their location in the cell. Another type of cell death, autophagy, can in some cases lead to cell death, while in other situations it acts as a cell survival mechanism. The present review considered the characteristics of the expression of HSPs of different molecular weights in CMCs in myocardial damage caused by heart failure, as well as their role in the realization of certain types of regulated cell death.

Thiol functionalised gold nanoparticles loaded with methotrexate for cancer treatment: From synthesis to in vitro studies on neuroblastoma cell lines.

HYPOTHESIS: Colloidal gold nanoparticles (AuNPs) functionalised with hydrophilic thiols can be used as drug delivery probes, thanks to their small size and hydrophilic character. AuNPs possess unique properties for their use in nanomedicine, especially in cancer treatment, as diagnostics and therapeutic tools. EXPERIMENTS: Thiol functionalised AuNPs were synthesised and loaded with methotrexate (MTX). Spectroscopic and morphostructural characterisations evidenced the stability of the colloids upon interaction with MTX. Solid state (GISAXS, GIWAXS, FESEM, TEM, FTIR-ATR, XPS) and dispersed phase (UV-Vis, DLS, ζ-potential, NMR, SAXS) experiments allowed to understand structure-properties correlations. The nanoconjugate was tested in vitro (MTT assays) against two neuroblastoma cell lines: SNJKP and IMR5 with overexpressed n-Myc. FINDINGS: Molar drug encapsulation efficiency was optimised to be >70%. A non-covalent interaction between the π system and the carboxylate moiety belonging to MTX and the charged aminic group of one of the thiols was found. The MTX loading slightly decreased the structural order of the system and increased the distance between the AuNPs. Free AuNPs showed no cytotoxicity whereas the AuNPs-MTX nanoconjugate had a more potent effect when compared to free MTX. The active role of AuNPs was evidenced by permeation studies: an improvement on penetration of the drug inside cells was evidenced.

Combined inhibition of polyamine metabolism and eIF5A hypusination suppresses colorectal cancer growth through a converging effect on MYC translation.

A key mechanism driving colorectal cancer (CRC) development is the upregulation of MYC and its targets, including ornithine decarboxylase (ODC), a master regulator of polyamine metabolism. Elevated polyamines promote tumorigenesis in part by activating DHPS-mediated hypusination of the translation factor eIF5A, thereby inducing MYC biosynthesis. Thus, MYC, ODC and eIF5A orchestrate a positive feedback loop that represents an attractive therapeutic target for CRC therapy. Here we show that combined inhibition of ODC and eIF5A induces a synergistic antitumor response in CRC cells, leading to MYC suppression. We found that genes of the polyamine biosynthesis and hypusination pathways are significantly upregulated in colorectal cancer patients and that inhibition of ODC or DHPS alone limits CRC cell proliferation through a cytostatic mechanism, while combined ODC and DHPS/eIF5A blockade induces a synergistic inhibition, accompanied to apoptotic cell death in vitro and in mouse models of CRC and FAP. Mechanistically, we found that this dual treatment causes complete inhibition of MYC biosynthesis in a bimodal fashion, by preventing translational elongation and initiation. Together, these data illustrate a novel strategy for CRC treatment, based on the combined suppression of ODC and eIF5A, which holds promise for the treatment of CRC.

An Undefined Interaction between Polyamines and Heat Shock Proteins Leads to Cellular Protection in Plasmodium falciparum and Proliferating Cells in Various Organisms.

Environmental stimuli can distress the internal reaction of cells and their normal function. To react promptly to sudden environmental changes, a cascade of heat shock proteins (Hsps) functions to protect and act as housekeepers inside the cells. In parallel to the heat shock response, the metabolic polyamine (PA) status changes. Here, we discuss possible ways of putative interactions between Hsps and polyamines in a wide lineage of eukaryotic model organisms with a particular focus on parasitic protozoa such as Plasmodium falciparum (P. falciparum). The supposed interaction between polyamines and Hsps may protect the parasite from the sudden change in temperature during transmission from the female Anopheles mosquito to a human host. Recent experiments performed with the spermidine mimetic inhibitor 15-deoxyspergualine in Plasmodium in vitro cultures show that the drug binds to the C-terminal EEVD motif of Hsp70. This leads to inhibition of protein biosynthesis caused by prevention of eIF5A2 phosphorylation and eukaryotic initiation factor 5A (eIF5A) modification. These observations provide further evidence that PAs are involved in the regulation of protein biosynthesis of Hsps to achieve a protective effect for the parasite during transmission.

Acidic Shift of Optimum pH of Bovine Serum Amine Oxidase upon Immobilization onto Nanostructured Ferric Tannates.

Protein-nanoparticle hybrids represent entities characterized by emerging biological properties that can significantly differ from those of the parent components. Herein, bovine serum amine oxidase (i.e., BSAO) was immobilized onto a magnetic nanomaterial constituted of surface active maghemite nanoparticles (i.e., SAMNs, the core), surface-modified with tannic acid (i.e., TA, the shell), to produce a biologically active ternary hybrid (i.e., SAMN@TA@BSAO). In comparison with the native enzyme, the secondary structure of the immobilized BSAO responded to pH variations sensitively, resulting in a shift of its optimum activity from pH 7.2 to 5.0. Conversely, the native enzyme structure was not influenced by pH and its activity was affected at pH 5.0, i.e., in correspondence with the best performances of SAMN@TA@BSAO. Thus, an extensive NMR study was dedicated to the structure-function relationship of native BSAO, confirming that its low activity below pH 6.0 was ascribable to minimal structural modifications not detected by circular dichroism. The generation of cytotoxic products, such as aldehydes and H2O2, by the catalytic activity of SAMN@TA@BSAO on polyamine oxidation is envisaged as smart nanotherapy for tumor cells. The present study supports protein-nanoparticle conjugation as a key for the modulation of biological functions.

Bovine Serum Amine Oxidase and Polyamine Analogues: Chemical Synthesis and Biological Evaluation Integrated with Molecular Docking and 3-D QSAR Studies.

Natural polyamines (PAs) are key players in cellular homeostasis by regulating cell growth and proliferation. Several observations highlight that PAs are also implicated in pathways regulating cell death. Indeed, the PA accumulation cytotoxic effect, maximized with the use of bovine serum amine oxidase (BSAO) enzyme, represents a valuable strategy against tumor progression. In the present study, along with the design, synthesis, and biological evaluation of a series of new spermine (Spm) analogues (1-23), a mixed structure-based (SB) and ligand-based (LB) protocol was applied. Binding modes of BSAO-PA modeled complexes led to clarify electrostatic and steric features likely affecting the BSAO-PA biochemical kinetics. LB and SB three-dimensional quantitative structure-activity relationship (Py-CoMFA and Py-ComBinE) models were developed by means of the 3d-qsar.com portal, and their analysis represents a strong basis for future design and synthesis of PA BSAO substrates for potential application in oxidative stress-induced chemotherapy.

Anticancer Cytotoxic Activity of Bispidine Derivatives Associated with the Increasing Catabolism of Polyamines.

Polyamine (PA) catabolism is often reduced in cancer cells. The activation of this metabolic pathway produces cytotoxic substances that might cause apoptosis in cancer cells. Chemical compounds able to restore the level of PA catabolism in tumors could become potential antineoplastic agents. The search for activators of PA catabolism among bicyclononan-9-ones is a promising strategy for drug development. The aim of the study was to evaluate the biological activity of new 3,7-diazabicyclo[3.3.1]nonan-9-one derivatives that have antiproliferative properties by accelerating PA catabolism. Eight bispidine derivatives were synthetized and demonstrated the ability to activate PA catabolism in regenerating rat liver homogenates. However, only three of them demonstrated a potent ability to decrease the viability of cancer cells in the MTT assay. Compounds 4c and 4e could induce apoptosis more effectively in cancer HepG2 cells rather than in normal WI-38 fibroblasts. The lead compound 4e could significantly enhance cancer cell death, but not the death of normal cells if PAs were added to the cell culture media. Thus, the bispidine derivative 4e 3-(3-methoxypropyl)-7-[3-(1H-piperazin-1-yl)ethyl]-3,7-diazabicyclo[3.3.1]nonane could become a potential anticancer drug substance whose mechanism relies on the induction of PA catabolism in cancer cells.

Pilot Investigation on p75ICD Expression in Laryngeal Squamous Cell Carcinoma.

We investigated the p75 Neurotrophin Receptor (p75NTR) expression and cleavage product p75NTR Intracellular Domain (p75ICD) as potential oncogenic and metastatic markers in human Laryngeal Squamous Cell Carcinoma (LSCC). p75NTR is highly expressed in Cancer Stem Cells (CSCs) of the laryngeal epithelia and it has been proposed as a marker for stemness, cell migration, and chemo-resistance in different squamous carcinomas. To investigate the clinical significance of p75NTR cleavage products in solid tumors, full-length and cleaved p75NTR expression was analyzed in laryngeal primary tumors from different-stage LSCC patients, diagnosed at the Policlinico Umberto I Hospital. Molecular and histological techniques were used to detect the expressions of p75NTR and p75ICD, and ATP Binding Cassette Subfamily G Member 2 (ABCG2), a CSC marker. We found regulated p75NTR cleavage during squamous epithelial tumor progression and tissue invasion. Our preliminary investigation suggests p75ICD expression and localization as possible features of tumorigenesis and metastaticity. Its co-localization with ABCG2 in squamous cells in the parenchyma invaded by the tumor formation allows us to hypothesize p75NTR and p75ICD roles in tumor invasion and CSC spreading in LSCC patients. These data might represent a starting point for a comprehensive analysis of p75NTR cleavage and of its clinical relevance as a potential molecular LSCC signature, possibly helping diagnosis, and improving prognosis and personalized therapy.

Alcohol and Head and Neck Cancer: Updates on the Role of Oxidative Stress, Genetic, Epigenetics, Oral Microbiota, Antioxidants, and Alkylating Agents.

Head and neck cancer (HNC) concerns more than 890,000 patients worldwide annually and is associated with the advanced stage at presentation and heavy outcomes. Alcohol drinking, together with tobacco smoking, and human papillomavirus infection are the main recognized risk factors. The tumorigenesis of HNC represents an intricate sequential process that implicates a gradual acquisition of genetic and epigenetics alterations targeting crucial pathways regulating cell growth, motility, and stromal interactions. Tumor microenvironment and growth factors also play a major role in HNC. Alcohol toxicity is caused both directly by ethanol and indirectly by its metabolic products, with the involvement of the oral microbiota and oxidative stress; alcohol might enhance the exposure of epithelial cells to carcinogens, causing epigenetic modifications, DNA damage, and inaccurate DNA repair with the formation of DNA adducts. Long-term markers of alcohol consumption, especially those detected in the hair, may provide crucial information on the real alcohol drinking of HNC patients. Strategies for prevention could include food supplements as polyphenols, and alkylating drugs as therapy that play a key role in HNC management. Indeed, polyphenols throughout their antioxidant and anti-inflammatory actions may counteract or limit the toxic effect of alcohol whereas alkylating agents inhibiting cancer cells' growth could reduce the carcinogenic damage induced by alcohol. Despite the established association between alcohol and HNC, a concerning pattern of alcohol consumption in survivors of HNC has been shown. It is of primary importance to increase the awareness of cancer risks associated with alcohol consumption, both in oncologic patients and the general population, to provide advice for reducing HNC prevalence and complications.

Hypusinated EIF5A as a feasible drug target for Advanced Medicinal Therapies in the treatment of pathogenic parasites and therapy-resistant tumors.

Cancer drug resistance, in particular in advanced stages such as metastasis and invasion is an emerging problem. Moreover, drug resistance of parasites causing poverty-related diseases is an enormous, global challenge for drug development in the future. To circumvent this problem of increasing resistance, the development of either novel small compounds or Advanced Medicinal Therapies have to be fostered. Polyamines have many fundamental cellular functions like DNA stabilization, protein translation, ion channel regulation, autophagy, apoptosis and mostly important, cell proliferation. Consequently, many antiproliferative drugs can be commonly administered either in cancer therapy or for the treatment of pathogenic parasites. Most important for cell proliferation is the triamine spermidine, since it is an important substrate in the biosynthesis of the posttranslational modification hypusine in eukaryotic initiation factor 5A (EIF5A). To date, no small compound has been identified that directly inhibits the precursor protein EIF5A. Moreover, only a few small molecule inhibitors of the two biosynthetic enzymes, i.e. deoxyhypusine synthase (DHS) and deoxyhypusine hydroxylase (DOHH) have been functionally characterized. However, it is evident that only some of the compounds have been applied in translational approaches, i.e. in murine models to analyze the function of this modified protein in cell proliferation. In recent years, the pharmaceutical industry shifted from small molecules beyond traditional pharmacology to new tools and methods to treat disorders involving signaling deregulation. In this review, we evaluate translational approaches on inhibition of EIF5A hypusination in pathogenic parasites and therapy-resistant tumors and discuss its feasibility for an application in Advanced Medicinal Therapies.

Urinary Ethyl Glucuronide for the Assessment of Alcohol Consumption During Pregnancy: Comparison between Biochemical Data and Screening Questionnaires.

BACKGROUND: Ethyl glucuronide (EtG) is a metabolite of ethanol used as a marker of alcohol drinking and is identified in urine. Gestational alcohol drinking harms the fetus, so disclosing any form of use and abuse of this substance during pregnancy is crucial. Many discovery methods have been planned to overcome this question, including using screening questionnaires as the AUDIT-C, T-ACE/TACER-3, and TWEAK. AIM: The aim and novelties of this study were to compare biochemical data from urinary EtG assays (cut-off 100 ng/mL for risking drinking behavior) with the outcome of questionnaires and of a food diary routinely used in our hospital; moreover, for the first time, we analyzed in pregnant women the EtG values normalized by the amount of creatinine excreted according to methods previously established. METHODS: Random urine samples were collected from 309 pregnant women immediately after being interviewed. EtG was quantified using an enzyme immunoassay, and urinary creatinine was assessed using an enzymatic colorimetric method. Women who had not exhaustively answered one of the questionnaires or refused to provide urine samples were excluded. Finally, 309 women were considered for this study. Urine creatinine measurements were performed to determine if urine dilution might have resulted in false negatives in the challenge study. In order to accomplish this objective, as urinary creatinine concentrations are, on average, approximately 1 mg/mL, we used a normalized value of 100 ng EtG/mg Creatinine. RESULTS: Our data show that 20.4% of the pregnant women in the study were over the established normalized cut-off value. Poor to null concordance (unweighted k < 0.2) was found between EtG data and the screening interviews showed, on average, lower levels of alcohol consumption. CONCLUSION: This study provides evidence that the assessment of maternal alcohol consumption during pregnancy, only indirectly estimated with questionnaires and food diary, can produce misleading results.

Translational control of polyamine metabolism by CNBP is required for Drosophila locomotor function.

Microsatellite expansions of CCTG repeats in the cellular nucleic acid-binding protein (CNBP) gene leads to accumulation of toxic RNA and have been associated with myotonic dystrophy type 2 (DM2). However, it is still unclear whether the dystrophic phenotype is also linked to CNBP decrease, a conserved CCHC-type zinc finger RNA-binding protein that regulates translation and is required for mammalian development. Here, we show that depletion of Drosophila CNBP in muscles causes ageing-dependent locomotor defects that are correlated with impaired polyamine metabolism. We demonstrate that the levels of ornithine decarboxylase (ODC) and polyamines are significantly reduced upon dCNBP depletion. Of note, we show a reduction of the CNBP-polyamine axis in muscles from DM2 patients. Mechanistically, we provide evidence that dCNBP controls polyamine metabolism through binding dOdc mRNA and regulating its translation. Remarkably, the locomotor defect of dCNBP-deficient flies is rescued by either polyamine supplementation or dOdc1 overexpression. We suggest that this dCNBP function is evolutionarily conserved in vertebrates with relevant implications for CNBP-related pathophysiological conditions.

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.

Anti-inflammatory role of curcumin in retinal disorders (Review).

Curcumin [1,7-bis-(4-hydroxy-3-methoxyphenyl)-hepta-1,6-diene-3,5-dione], the main component of turmeric (Curcuma longa, a flowering plant of the ginger family, Zingiberaceae), is known to possess different pharmacological activities, particularly anti-inflammatory and antioxidant properties. Since an underlying inflammatory process exists in several ocular conditions, such as anterior uveitis, glaucoma, age-related macular degeneration (AMD) and diabetic retinopathy (DR), the aim of the present review was to summarize the pleiotropic effects exerted by this molecule, focusing in particular on its beneficial role in retinal diseases. The anti-inflammatory activity of curcumin has also been described in numerous systemic inflammatory pathologies and tumors. Specifically, the biological, pharmaceutical and nutraceutical properties of curcumin are associated with its ability to downregulate the expression of the following genes: IκBα, cyclooxygenase 2, prostaglandin E2, interleukin (IL)-1, IL-6, IL-8 and tumor necrosis factor-α. According to this finding, curcumin may be useful in the treatment of some retinal disorders. In DR, proliferative vitreoretinopathy and AMD, beneficial effects have been observed following treatment with curcumin, including slowing down of the inflammatory process. Despite the aforementioned evidence, the main disadvantage of this substance is that it possesses a low solubility, as well as poor oral bioavailability due to its reduced absorption, rapid metabolism and rapid elimination. Therefore, several curcumin analogues have been synthesized and tested over the years, in order to improve the possible obtainable therapeutic effects. The purpose of the present review was to identify new aspects that could guide future research on this important traditional medicine, which is a well-tolerated natural product, and is widely considered safe and economical.

BCL2­regulated apoptotic process in myocardial ischemia­reperfusion injury (Review).

The leading cause of death in developed countries is cardiovascular disease, where coronary heart disease is the main cause of death. Myocardial reperfusion is the most significant method to prevent cell death after ischemia. However, restoration of blood flow may paradoxically lead to myocardial ischemia­reperfusion injury (MI/RI) accompanied by metabolic disturbances and cardiomyocyte death. As the myocardium has an extremely limited ability to regenerate, the mechanisms of regulated cell death, including apoptosis, are the most significant for contemporary research due to their reversibility. BCL2 is a key anti­apoptotic protein. There are several signaling pathways and compounds regulating BCL2, including PI3K/AKT and MEK1/ERK1/2, JAK2/STAT3, endothelial nitric oxide synthase, PTEN, cardiac ankyrin repeat protein and microRNA, which can serve as targets for modern methods of cardioprotective therapy inhibiting intrinsic apoptosis and saving viable cardiomyocytes after MI/RI. The present review considers the mechanisms of Bcl2­regulated apoptosis in the development and treatment of MI/RI.

Blockade of EIF5A hypusination limits colorectal cancer growth by inhibiting MYC elongation.

Eukaryotic Translation Initiation Factor 5A (EIF5A) is a translation factor regulated by hypusination, a unique posttranslational modification catalyzed by deoxyhypusine synthetase (DHPS) and deoxyhypusine hydroxylase (DOHH) starting from the polyamine spermidine. Emerging data are showing that hypusinated EIF5A regulates key cellular processes such as autophagy, senescence, polyamine homeostasis, energy metabolism, and plays a role in cancer. However, the effects of EIF5A inhibition in preclinical cancer models, the mechanism of action, and specific translational targets are still poorly understood. We show here that hypusinated EIF5A promotes growth of colorectal cancer (CRC) cells by directly regulating MYC biosynthesis at specific pausing motifs. Inhibition of EIF5A hypusination with the DHPS inhibitor GC7 or through lentiviral-mediated knockdown of DHPS or EIF5A reduces the growth of various CRC cells. Multiplex gene expression analysis reveals that inhibition of hypusination impairs the expression of transcripts regulated by MYC, suggesting the involvement of this oncogene in the observed effect. Indeed, we demonstrate that EIF5A regulates MYC elongation without affecting its mRNA content or protein stability, by alleviating ribosome stalling at five distinct pausing motifs in MYC CDS. Of note, we show that blockade of the hypusination axis elicits a remarkable growth inhibitory effect in preclinical models of CRC and significantly reduces the size of polyps in APCMin/+ mice, a model of human familial adenomatous polyposis (FAP). Together, these data illustrate an unprecedented mechanism, whereby the tumor-promoting properties of hypusinated EIF5A are linked to its ability to regulate MYC elongation and provide a rationale for the use of DHPS/EIF5A inhibitors in CRC therapy.

Nanotechnology-Based Strategies to Develop New Anticancer Therapies.

The blooming of nanotechnology has made available a limitless landscape of solutions responding to crucial issues in many fields and, nowadays, a wide choice of nanotechnology-based strategies can be adopted to circumvent the limitations of conventional therapies for cancer. Herein, the current stage of nanotechnological applications for cancer management is summarized encompassing the core nanomaterials as well as the available chemical-physical approaches for their surface functionalization and drug ligands as possible therapeutic agents. The use of nanomaterials as vehicles to delivery various therapeutic substances is reported emphasizing advantages, such as the high drug loading, the enhancement of the pay-load half-life and bioavailability. Particular attention was dedicated to highlight the importance of nanomaterial intrinsic features. Indeed, the ability of combining the properties of the transported drug with the ones of the nano-sized carrier can lead to multifunctional theranostic tools. In this view, fluorescence of carbon quantum dots, optical properties of gold nanoparticle and superparamagnetism of iron oxide nanoparticles, are fundamental examples. Furthermore, smart anticancer devices can be developed by conjugating enzymes to nanoparticles, as in the case of bovine serum amine oxidase (BSAO) and gold nanoparticles. The present review is aimed at providing an overall vision on nanotechnological strategies to face the threat of human cancer, comprising opportunities and challenges.

Phenformin Inhibits Hedgehog-Dependent Tumor Growth through a Complex I-Independent Redox/Corepressor Module.

The antidiabetic drug phenformin displays potent anticancer activity in different tumors, but its mechanism of action remains elusive. Using Shh medulloblastoma as model, we show here that at clinically relevant concentrations, phenformin elicits a significant therapeutic effect through a redox-dependent but complex I-independent mechanism. Phenformin inhibits mitochondrial glycerophosphate dehydrogenase (mGPD), a component of the glycerophosphate shuttle, and causes elevations of intracellular NADH content. Inhibition of mGPD mimics phenformin action and promotes an association between corepressor CtBP2 and Gli1, thereby inhibiting Hh transcriptional output and tumor growth. Because ablation of CtBP2 abrogates the therapeutic effect of phenformin in mice, these data illustrate a biguanide-mediated redox/corepressor interplay, which may represent a relevant target for tumor therapy.

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).