Search results for: phenolic antioxidant

Authors: Zuzana Tatarkova, Ivana Pilchova, Michal Cibulka, Martin Kolisek, Peter Racay, Peter Kaplan

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Introduction: Normal functioning of mitochondria is crucial for cardiac performance. Mitochondria undergo mitophagy and biogenesis, and mitochondrial proteins are subject to extensive post-translational modifications. The state of mitochondrial homeostasis reflects overall cellular fitness and longevity. Perturbed mitochondria produce less ATP, release greater amounts of reactive molecules, and are more prone to apoptosis. Therefore mitochondrial turnover is an integral aspect of quality control in which dysfunctional mitochondria are selectively eliminated through mitophagy. Currently, the progressive deterioration of physiological functions is seen as accumulation of modified/damaged proteins with limiting regenerative ability and disturbance of such affected protein-protein communication throughout aging in myocardial cells. Methodologies: For our study was used immunohistochemistry, biochemical methods: spectrophotometry, western blotting, immunodetection as well as more sophisticated 2D electrophoresis and mass spectrometry for evaluation protein-protein interactions and specific post-translational modification. Results and Discussion: Mitochondrial stress response to reactive species was evaluated as electron transport chain (ETC) complexes, redox-active molecules, and their possible communication. Protein-protein interactions revealed a strong linkage between age and ETC protein subunits. Redox state was strongly affected in senescent mitochondria with shift in favor of more pro-oxidizing condition within cardiomyocytes. Acute myocardial ischemia and ischemia-reperfusion (IR) injury affected ETC complexes I, II and IV with no change in complex III. Ischemia induced decrease in total antioxidant capacity, MnSOD, GSH and catalase activity with recovery in some extent during reperfusion. While MnSOD protein content was higher in IR group, activity returned to 95% of control. Nitric oxide is one of the biological molecules that can out compete MnSOD for superoxide and produce peroxynitrite. This process is faster than dismutation and led to the 10-fold higher production of nitrotyrosine after IR injury in adult with higher protection in senescent ones. 2D protein profiling revealed 140 mitochondrial proteins, 12 of them with significant changes after IR injury and 36 individual nitrotyrosine-modified proteins further identified by mass spectrometry. Linking these two groups, 5 proteins were altered after IR as well as nitrated, but only one showed massive nitration per lowering content of protein after IR injury in adult. Conclusions: Senescent cells have greater proportion of protein content, which might be modulated by several post-translational modifications. If these protein modifications are connected to functional consequences and protein-protein interactions are revealed, link may lead to the solution. Assume all together, dysfunctional proteostasis can play a causative role and restoration of protein homeostasis machinery is protective against aging and possibly age-related disorders. This work was supported by the project VEGA 1/0018/18 and by project 'Competence Center for Research and Development in the field of Diagnostics and Therapy of Oncological diseases', ITMS: 26220220153, co-financed from EU sources.

Keywords: aging heart, mitochondria, proteomics, redox state

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Authors: Kyriaki Hatziagapiou, Konstantinos Bethanis, Olti Nikola, Elias Christoforides, Eleni Koniari, Eleni Kakouri, George Lambrou, Christina Kanaka-Gantenbein

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Glioblastoma multiforme (GBM) remains a serious health challenge, as current therapeutic modalities continue to yield unsatisfactory results, with the average survival rarely exceeding 1-2 years. Natural compounds still provide some of the most promising approaches for discovering new drugs. The non-psychotropic cannabidiol (CBD) deriving from Cannabis sativa L. provides such promise. CBD is endowed with anticancer, antioxidant, and genoprotective properties as established in vitro and in in vivo experiments. CBD’s selectivity towards cancer cells and its safe profile suggest its usage in cancer therapies. However, the bioavailability of oral CBD is low due to poor aqueous solubility, erratic gastrointestinal absorption, and significant first-pass metabolism, hampering its therapeutic potential and resulting in a variable pharmacokinetic profile. In this context, CBD can take great advantage of nanomedicine-based formulation strategies. Cyclodextrins (CDs) are cyclic oligosaccharides used in the pharmaceutical industry to incorporate apolar molecules inside their hydrophobic cavity, increasing their stability, water solubility, and bioavailability or decreasing their side effects. CBD-inclusion complexes with CDs could be a good strategy to improve its properties, like solubility and stability to harness its full therapeutic potential. The current research aims to study the potential cytotoxic effect of CBD and CBD-CDs complexes CBD-RMβCD (randomly methylated β-cyclodextrin) and CBD-HPβCD (hydroxypropyl-b-CD) on the A172 glioblastoma cell line. CBD is diluted in 10% DMSO, and CBD/CDs solutions are prepared by mixing solid CBD, solid CDs, and dH2O. For the biological assays, A172 cells are incubated at a range of concentrations of CBD, CBD-RMβCD and CBD-HPβCD, RMβCD, and HPβCD (0,03125-4 mg/ml) at 24, 48, and 72 hours. Analysis of cell viability after incubation with the compounds is performed with Alamar Blue viability assay. CBD’s dilution to DMSO 10% was inadequate, as crystals are observed; thus cytotoxicity experiments are not assessed. CBD’s solubility is enhanced in the presence of both CDs. CBD/CDs exert significant cytotoxicity in a dose and time-dependent manner (p < 0.005 for exposed cells to any concentration at 48, 72, and 96 hours versus cells not exposed); as their concentration and time of exposure increases, the reduction of resazurin to resofurin decreases, indicating a reduction in cell viability. The cytotoxic effect is more pronounced in cells exposed to CBD-HPβCD for all concentrations and time-points. RMβCD and HPβCD at the highest concentration of 4 mg/ml also exerted antitumor action per se since manifesting cell growth inhibition. The results of our study could afford the basis of research regarding the use of natural products and their inclusion complexes as anticancer agents and the shift to targeted therapy with higher efficacy and limited toxicity. Acknowledgments: The research is partly funded by ΙΚΥ (State Scholarships Foundation) – Post-doc Scholarships-Partnership Agreement 2014-2020.

Keywords: cannabidiol, cyclodextrins, glioblastoma, hydroxypropyl-b-Cyclodextrin, randomly-methylated-β-cyclodextrin

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Authors: Chuanxiang Cheng, Tiantian Min, Jin Yue

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Antimicrobial active packaging enables extensive biological effects to improve food safety. However, the efficacy of antimicrobial packaging hinges on factors including the diffusion rate of the active agent toward the food surface, the initial content in the antimicrobial agent, and the targeted food shelf life. Among the possibilities of antimicrobial packaging design, an interesting approach involves the incorporation of volatile antimicrobial agents into the packaging material. In this case, the necessity for direct contact between the active packaging material and the food surface is mitigated, as the antimicrobial agent exerts its action through the packaging headspace atmosphere towards the food surface. However, it still remains difficult to achieve controlled and precise release of bioactive compounds to the specific target location with required quantity in food packaging applications. Remarkably, the development of stimuli-responsive materials for electrospinning has introduced the possibility of achieving controlled release of active agents under specific conditions, thereby yielding enduring biological effects. Relative humidity (RH) for the storage of food categories such as meat and aquatic products typically exceeds 90%. Consequently, high RH can be used as an abiotic trigger for the release of active agents to prevent microbial growth. Hence, a novel RH - responsive polyvinyl alcohol/chitosan (PVA/CS) composite nanofibrous film incorporated with 4-terpineol/β-cyclodextrin inclusion complexes (4-TA@β-CD ICs) was engineered by electrospinning that can be deposited as a functional packaging materials. The characterization results showed the thermal stability of the films was enhanced after the incorporation due to the hydrogen bonds between ICs and polymers. Remarkably, the 4 wt% 4-TA@β-CD ICs/PVA/CS film exhibited enhanced crystallinity, moderate hydrophilic (Water contact angle of 81.53°), light barrier property (Transparency of 1.96%) and water resistance (Water vapor permeability of 3.17 g mm/m2 h kPa). Moreover, this film also showed optimized mechanical performance with a Young’s modulus of 11.33 MPa, a tensile strength of 19.99 MPa and an elongation at break of 4.44 %. Notably, the antioxidant and antibacterial properties of this packaging material were significantly improved. The film demonstrated the half-inhibitory concentrations (IC50) values of 87.74% and 85.11% for scavenging 2,2-diphenyl-1-picrylhydrazyl (DPPH) and 2, 2′-azinobis (3-ethylbenzothiazoline-6-sulfonic) (ABTS) free radicals, respectively, in addition to an inhibition efficiency of 65% against Shewanella putrefaciens, the characteristic bacteria in aquatic products. Most importantly, the film achieved controlled release of 4-TA under high 98% RH by inducing the plasticization of polymers caused by water molecules, swelling of polymer chains, and destruction of hydrogen bonds within the cyclodextrin inclusion complex. Consequently, low relative humidity is suitable for the preservation of nanofibrous film, while high humidity conditions typical in fresh food packaging environments effectively stimulated the release of active compounds in the film. This film with a long-term antimicrobial effect successfully extended the shelf life of Litopenaeus vannamei shrimp to 7 days at 4 °C. This attractive design could pave the way for the development of new food packaging materials.

Keywords: controlled release, electrospinning, nanofibrous film, relative humidity–responsive, shrimp preservation

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Authors: Mohammadjavad Sotoudeheian, Navid Farahmandian

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Kawasaki disease (KD) is the primary cause of acquired pediatric heart disease as an acute vasculitis. In children with prolonged fever, rash, and inflammation of the mucosa KD must be considered as a clinical diagnosis. There is a persuasive suggestion of immune-mediated damage as the pathophysiologic cascade of KD. For example, the invasion of cytotoxic T-cells supports a viral etiology and the inflammasome of the innate immune system is a critical component in the vasculitis formation in KD. Animal models of KD propose the cytokine profiles, such as increased IL-1 and GM-CSF, which cause vascular damage. CRP and IFN-γ elevated expression and the upregulation of IL-6, and IL-10 production are also described in previous studies. Untreated KD is a critical risk factor for coronary artery diseases and myocardial infarction. Vascular damage may encompass amplified T-cell activity. SMAD3 is an essential molecule in down-regulating T-cells and increasing expression of FoxP3. It has a critical effect in the differentiation of regulatory T-cells. The discrepancy of regulatory T-cells and pro-inflammatory Th17 has been studied in acute coronary syndrome during KD. However in the coronary artery damaged lymphocytes and IgA plasma cells are seen at the lesion locations, the major immune cells in the coronary lesions are monocytes/macrophages and neutrophils. These cells secrete TNF-α, and activates matrix metalloprotease (MMP)-9, reducing the integrity of vessels and prompting patients to arise aneurysm. MMPs can break down the components of the extracellular matrix and assist immune cell movement. IVIG as an effective form of treatment clarified the role of the immune system, which may target pathogenic antigens and regulate cytokine production. Several reports have revealed that in the coronary arteries, high expression of MMP-9 in monocyte/macrophage results in pathologic cascades. Curcumin is a potent antioxidant and anti-inflammatory molecule. Curcumin decreases the production of reactive oxygen and nitrogen species and inhibits transcription factors like AP-1 and NF-κB. Curcumin also contains the characteristics of inhibitory effects on MMPs, especially MMP-9. The upregulation of MMP-9 is an important cellular response. Curcumin treatment caused a reverse effect and down-regulates MMP-9 gene expression which may fund the anti-inflammatory effect. Curcumin inhibits MMP-9 expression via PKC and AMPK-dependent pathways in Human monocytes cells. Elevated expression and activity of MMP-9 are correlated with advanced vascular lesions. AMPK controls lipid metabolism and oxidation, and protein synthesis. AMPK is also necessary for the MMP-9 activity and THP-1 cell adhesion to endothelial cells. Curcumin was shown to inhibit the activation of AMPKα. Compound C (AMPK inhibitor) inhibits MMP-9 expression level. Therefore, through inactivating AMPKs and PKC, curcumin decreases the MMP-9 level, which results in inhibiting monocyte/macrophage differentiation. Compound C also suppress the phosphorylation of three major classes of MAP kinase signaling, suggesting that curcumin may suppress MMP-9 level by inactivation of MAPK pathways. MAPK cascades are activated to induce the expression of MMP-9. Curcumin inhibits MAPKs phosphorylation, which contributes to the down-regulation of MMP-9. This study demonstrated that the potential inhibitory properties of curcumin over MMP-9 lead to a therapeutic strategy to reduce the risk of coronary artery involvement during KD.

Keywords: MMP-9, coronary artery aneurysm, Kawasaki’s disease, curcumin, AMPK, immune system, NF-κB, MAPK

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Authors: Piacentini Diego, Della Rovere Federica, Fattorini Laura, Lanni Francesca, Cittadini Martina, Altamura Maria Maddalena, Falasca Giuseppina

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Plants have evolved sophisticated mechanisms to cope with environmental cues. Changes in intracellular content and distribution of phytohormones, such as the auxin indole-3-acetic acid (IAA), have been involved in morphogenic adaptation to environmental stresses. In addition to phytohormones, plants can rely on a plethora of small signal molecules able to promptly sense and transduce the stress signals, resulting in morpho/physiological responses thanks also to their capacity to modulate the levels/distribution/reception of most hormones. Among these signaling molecules, nitrogen monoxide (nitric oxide – NO) is a critical component in several plant acclimation strategies to both biotic and abiotic stresses. Depending on its levels, NO increases plant adaptation by enhancing the enzymatic or non-enzymatic antioxidant systems or by acting as a direct scavenger of reactive oxygen/nitrogen (ROS/RNS) species produced during the stress. In addition, exogenous applications of NO-specific donor compounds showed the involvement of the signal molecule in auxin metabolism, transport, and signaling, under both physiological and stress conditions. However, the complex mechanisms underlying NO action in interacting with phytohormones, such as auxins, during metal stress responses are still poorly understood and need to be better investigated. Emphasis must be placed on the response of the root system since it is the first plant organ system to be exposed to metal soil pollution. The monocot Oryza sativa L. (rice) has been chosen given its importance as a stable food for some 4 billion people worldwide. In addition, increasing evidence has shown that rice is often grown in contaminated paddy soils with high levels of heavy metal cadmium (Cd) and metalloid arsenic (As). The facility through which these metals are taken up by rice roots and transported to the aerial organs up to the edible caryopses makes rice one of the most relevant sources of these pollutants for humans. This study aimed to evaluate if NO has a mitigatory activity in the roots of rice seedlings against Cd or As toxicity and to understand if this activity requires interactions with auxin. Our results show that exogenous treatments with the NO-donor SNP alleviate the stress induced by Cd, but not by As, in in-vitro-grown rice seedlings through increased intracellular root NO levels. The damages induced by the pollutants include root growth inhibition, root histological alterations and ROS (H2O2, O2●ˉ), and RNS (ONOOˉ) production. Also, SNP treatments mitigate both the root increase in root IAA levels and the IAA alteration in distribution monitored by the OsDR5::GUS system due to the toxic metal exposure. Notably, the SNP-induced mitigation of the IAA homeostasis altered by the pollutants does not involve changes in the expression of OsYUCCA1 and ASA2 IAA-biosynthetic genes. Taken together, the results highlight a mitigating role of NO in the rice root system, which is pollutant-specific, and involves the interaction of the signal molecule with both IAA and brassinosteroids at different (i.e., transport, levels, distribution) and multiple levels (i.e., transcriptional/post-translational levels). The research is supported by Progetti Ateneo Sapienza University of Rome, grant number: RG120172B773D1FF

Keywords: arsenic, auxin, cadmium, nitric oxide, rice, root system

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Authors: Marcela Parra-Vargas, Ana Sandoval-Rodriguez, Roberto Rodriguez-Echevarria, Jose Dominguez-Rosales, Juan Armendariz-Borunda

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Non-alcoholic fatty liver disease (NAFLD) is characterized by an excess of hepatic lipids, and it is to author’s best knowledge, the most prevalent chronic liver disorder. Anthocyanin-rich food consumption is linked to health benefits in metabolic disorders associated with obesity and NAFLD, although the precise functional role of anthocyanidin delphinidin (Dp) has yet to be established. The aim of this study was to investigate the effect of the Dp in NAFLD metabolic alterations by evaluating prevention or amelioration of hepatic lipid accumulation, as well as molecular mechanisms in two experimental obesity-related models of NALFD. In vitro: HepG2 cells were incubated with sodium palmitate (PA, 1 mM) to induce lipotoxic damage, and concomitantly treated with Dp (180 uM) for 24 h. Subsequently, total lipid accumulation was measured by colorimetric staining with Oil Red O, and total intrahepatic triglycerides were determined by an enzymatic assay. To assess molecular mechanisms, cells were pre-treated with PA for 24 h and then exposed to Dp for 1 h. In vivo: four-week-old male C57BL/6Nhsd mice were allocated in two main groups. Mice were fed with standard diet (control) or high-fat and high-carbohydrate diet (45% fat, HFD) for 16 wk to induce NAFLD. Then HFD was divided into subgroups: one treated orally with Dp (15 mg/kg bw, HFD-Dp) every day for 4 wk, while HFD group treated with vehicle (DMSO). Weight and fasting glucose were recorded weekly, while dietary ingestion was measured daily. Insulin tolerance test was performed at the end of treatment. Liver histology was evaluated with H&E and Masson’s trichrome stain. RT-PCR was used to evaluate gene expression and Western Blot to determine levels of protein in both experimental models. Parametric data were analyzed with one-way ANOVA and Tukey’s post-hoc test. Kruskal-Wallis and Mann-Whitney U test for non-parametric data, and P < 0.5 were considered significant. Dp prevented hepatic lipid accumulation by PA in HepG2 hepatocytes. Furthermore, Dp down-regulated gene expression of SREBP1c, FAS, and CPT1a without modifying AMPK phosphorylation levels. In vivo, Dp oral administration did not ameliorate lipid metabolic alterations raised by HFD. Adiposity, dietary ingestion, fasting glucose, and insulin sensitivity after Dp treatment remained similar to HFD group. Histological analysis showed hepatic damage in HFD groups and no differences between HFD and HFD-Dp groups were found. Hepatic gene expression of ACC and FAS were not altered by HFD. SREBP1c was similar in both HFD and HFD-Dp groups. No significant changes were observed in SREBP1c, ACC, and FAS adipose tissue gene expression by HFD or Dp treatment. Additionally, immunoblotting analysis revealed no changes in pathway SIRT1-LKB-AMPK and PPAR alpha by both HFD groups compared to control. In conclusion, the antioxidant Dp may provoke beneficial effects in the prevention of hepatic lipid accumulation. Nevertheless, the oral dose administrated in mice that simulated the total intake of anthocyanins consumed daily by humans has no effect as a treatment on hepatic lipid metabolic alterations and histological abnormalities associated with exposure to chronic HFD. A healthy lifestyle with regular intake of antioxidants such as anthocyanins may prevent metabolic alterations in NAFLD.

Keywords: anthocyanins, antioxidants, delphinidin, non-alcoholic fatty liver disease, obesity

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Authors: Siddhant Yadav, Rylea Ranum, Hannah Alberts, Abdul Kalaiger, Brent Bauer, Ryan Hurt, Ann Vincent, Loren Toussaint, Sanjeev Nanda

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Background and significance: Fibromyalgia (FM) is a chronic pain disorder also characterized by chronic fatigue, morning stiffness, sleep, and cognitive symptoms, psychological disturbances (anxiety, depression), and is comorbid with multiple medical and psychiatric conditions. It has an incidence of 2-4% in the general population and is reported more commonly in women. Oxidative stress and inflammation are thought to contribute to pain in patients with FM, and the adoption of an antioxidant/anti-inflammatory diet has been suggested as a modality to alleviate symptoms. The aim of this systematic review was to evaluate the efficacy of specialized diets (ketogenic, gluten free, Mediterranean, and low carbohydrate) in improving FM symptoms. Methodology: A comprehensive search of the following databases from inception to July 15th, 2021, was conducted: Ovid MEDLINE and Epub ahead of print, in-process and other non-indexed citations and daily, Ovid Embase, Ovid EBM reviews, Cochrane central register of controlled trials, EBSCO host CINAHL with full text, Elsevier Scopus, website and citation index, web of science emerging sources citation and clinicaltrials.gov. We included randomized controlled trials, non-randomized experimental studies, cross-sectional studies, cohort studies, case series, and case reports in adults with fibromyalgia. The risk of bias was assessed with the Agency for Health Care Research and Quality designed, specific recommended criteria (AHRQ). Results: Thirteen studies were eligible for inclusion. This included a total of 761 participants. Twelve out of the 13 studies reported improvement in widespread body pain, joint stiffness, sleeping pattern, mood, and gastrointestinal symptoms, and one study reported no changes in symptomatology in patients with FM on specialized diets. None of the studies showed the worsening of symptoms associated with a specific diet. Most of the patient population was female, with the mean age at which fibromyalgia was diagnosed being 48.12 years. Improvement in symptoms was reported by the patient's adhering to a gluten-free diet, raw vegan diet, tryptophan- and magnesium-enriched Mediterranean diet, aspartame- and msg- elimination diet, and specifically a Khorasan wheat diet. Risk of bias assessment noted that 6 studies had a low risk of bias (5 clinical trials and 1 case series), four studies had a moderate risk of bias, and 3 had a high risk of bias. In many of the studies, the allocation of treatment (diets) was not adequately concealed, and the researchers did not rule out any potential impact from a concurrent intervention or an unintended exposure that might have biased the results. On the other hand, there was a low risk of attrition bias in all the trials; all were conducted with an intention-to-treat, and the inclusion/exclusion criteria, exposures/interventions, and primary outcomes were valid, reliable, and implemented consistently across all study participants. Concluding statement: Patients with fibromyalgia who followed specialized diets experienced a variable degree of improvement in their widespread body pain. Improvement was also seen in stiffness, fatigue, moods, sleeping patterns, and gastrointestinal symptoms. Additionally, the majority of the patients also reported improvement in overall quality of life.

Keywords: fibromyalgia, specialized diet, vegan, gluten free, Mediterranean, systematic review

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Authors: Kuladip Jana

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Benzo(a)pyrene [B(a)P] is an environmental toxicant present mostly in cigarette smoke and car exhaust, is an aryl hydrocarbon receptor (AhR) ligand that exerts its toxic effects on both male and female reproductive systems. In this study, the effect of B(a)P at different doses (0.1, 0.25, 0.5, 1 and 5 mg /kg body weight) was studied on male reproductive system of rat. A significant decrease in cauda epididymal sperm count and motility along with the presence of sperm head abnormalities and altered epididymal and testicular histology were documented following B(a)P treatment. B(a)P treatment resulted apoptotic sperm cells as observed by TUNEL and Annexin V-PI assay with increased ROS, altered sperm mitochondrial membrane potential (ΔΨm) with a simultaneous decrease in the activity of antioxidant enzymes and GSH status. TUNEL positive apoptotic cells also observed in testis as well as isolated germ and Leydig cells following B(a)P exposure. Western Blot analysis revealed the activation of p38MAPK, cytosolic translocation of cytochrome-c, up-regulation of Bax and inducible nitric oxide synthase (iNOS) with cleavage of PARP and down-regulation of BCl2 in testis upon B(a)P treatment. The protein and mRNA levels of testicular key steroidogenesis regulatory proteins like StAR, cytochrome P450 IIA1 (CYPIIA1), 3β HSD, 17β HSD showed a significant decrease in a dose dependent manner while an increase in the expression of cytochrome P450 1A1 (CYP1A1), Aryl hydrocarbon Receptor (AhR), active caspase- 9 and caspase- 3 following B(a)P exposure. We conclude that exposure of benzo(a)pyrene caused testicular gamatogenic and steroidogenic disorders by induction of oxidative stress, inhibition of StAR and other steroidogenic enzymes along with activation of p38MAPK and initiated caspase-3 mediated germ and Leydig cell apoptosis.The possible protective role of naturally occurring phytochemicals against B(a)P induced testicular toxicity needs immediate consideration. Curcumin and resveratrol separately were found to protect against B(a)P induced germ cell apoptosis, and their combinatorial effect was more significant. Our present study in isolated testicular germ cell population from adult male Wistar rats, highlighted their synergistic protective effect against B(a)P induced germ cell apoptosis. Curcumin-resveratrol co-treatment decreased the expression of pro-apoptotic proteins like cleaved caspase 3,8,9, cleaved PARP, Apaf1, FasL, tBid. Curcumin-resveratrol co-treatment decreased Bax/Bcl2 ratio, mitochondria to cytosolic translocation of cytochrome c and activated the survival protein Akt. Curcumin-resveratrol decreased the expression of p53 dependent apoptotic genes like Fas, FasL, Bax, Bcl2, Apaf1.Curcumin-resveratrol co-treatment thus prevented B(a)P induced germ cell apoptosis. B(a)P induced testicular ROS generation and oxidative stress were significantly ameliorated with curcumin and resveratrol. Curcumin-resveratrol co-treatment prevented B(a)P induced nuclear translocation of AhR and CYP1A1 production. The combinatorial treatment significantly inhibited B(a)P induced ERK 1/2, p38 MAPK and JNK 1/2 activation. B(a)P treatment increased the expression of p53 and its phosphorylation (p53 ser 15). Curcumin-resveratrol co-treatment significantly decreased p53 level and its phosphorylation (p53 ser 15). The study concludes that curcumin-resveratrol synergistically modulated MAPKs and p53, prevented oxidative stress, regulated the expression of pro and anti-apoptotic proteins as well as the proteins involved in B(a)P metabolism thus protected germ cells from B(a)P induced apoptosis.

Keywords: benzo(a)pyrene, germ cell, apoptosis, oxidative stress, resveratrol, curcumin

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Authors: Kyriaki Hatziagapiou, Eleni Kakouri, Konstantinos Bethanis, Alexandra Nikola, Eleni Koniari, Charalabos Kanakis, Elias Christoforides, George Lambrou, Petros Tarantilis

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Medulloblastoma is a highly invasive tumour, as it tends to disseminate throughout the central nervous system early in its course. Despite the high 5-year-survival rate, a significant number of patients demonstrate serious long- or short-term sequelae (e.g., myelosuppression, endocrine dysfunction, cardiotoxicity, neurological deficits and cognitive impairment) and higher mortality rates, unrelated to the initial malignancy itself but rather to the aggressive treatment. A strong rationale exists for the use of Crocus sativus L (saffron) and its bioactive constituents (crocin, crocetin, safranal) as pharmaceutical agents, as they exert significant health-promoting properties. Crocins are water soluble carotenoids. Unlike other carotenoids, crocins are highly water-soluble compounds, with relatively low toxicity as they are not stored in adipose and liver tissues. Crocins have attracted wide attention as promising anti-cancer agents, due to their antioxidant, anti-inflammatory, and immunomodulatory effects, interference with transduction pathways implicated in tumorigenesis, angiogenesis, and metastasis (disruption of mitotic spindle assembly, inhibition of DNA topoisomerases, cell-cycle arrest, apoptosis or cell differentiation) and sensitization of cancer cells to radiotherapy and chemotherapy. The current research aimed to study the potential cytotoxic effect of crocins on TE671 medulloblastoma cell line, which may be useful in the optimization of existing and development of new therapeutic strategies. Crocins were extracted from stigmas of saffron in ultrasonic bath, using petroleum-ether, diethylether and methanol 70%v/v as solvents and the final extract was lyophilized. Identification of crocins according to high-performance liquid chromatography (HPLC) analysis was determined comparing the UV-vis spectra and the retention time (tR) of the peaks with literature data. For the biological assays crocin was diluted to nuclease and protease free water. TE671 cells were incubated with a range of concentrations of crocins (16, 8, 4, 2, 1, 0.5 and 0.25 mg/ml) for 24, 48, 72 and 96 hours. Analysis of cell viability after incubation with crocins was performed with Alamar Blue viability assay. The active ingredient of Alamar Blue, resazurin, is a blue, nontoxic, cell permeable compound virtually nonfluorescent. Upon entering cells, resazurin is reduced to a pink and fluorescent molecule, resorufin. Viable cells continuously convert resazurin to resorufin, generating a quantitative measure of viability. The colour of resorufin was quantified by measuring the absorbance of the solution at 600 nm with a spectrophotometer. HPLC analysis indicated that the most abundant crocins in our extract were trans-crocin-4 and trans-crocin-3. Crocins exerted significant cytotoxicity in a dose and time-dependent manner (p < 0.005 for exposed cells to any concentration at 48, 72 and 96 hours versus cells not exposed); as their concentration and time of exposure increased, the reduction of resazurin to resofurin decreased, indicating reduction in cell viability. IC50 values for each time point were calculated ~3.738, 1.725, 0.878 and 0.7566 mg/ml at 24, 48, 72 and 96 hours, respectively. The results of our study could afford the basis of research regarding the use of natural carotenoids as anticancer agents and the shift to targeted therapy with higher efficacy and limited toxicity. Acknowledgements: The research was funded by Fellowships of Excellence for Postgraduate Studies IKY-Siemens Programme.

Keywords: crocetin, crocin, medulloblastoma, saffron

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