N6-isopentenyladenosine inhibits aerobic glycolysis in glioblastoma cells by targeting PKM2 expression and activity.

Glioblastoma (GBM) is a primary tumor in the central nervous system with poor prognosis. It exhibits elevated glucose uptake and lactate production. This metabolic state of aerobic glycolysis is known as the Warburg effect. N6-isopentenyladenosine (iPA), a natural cytokine modified with an isopentenyl moiety derived from the mevalonate pathway, has well-established anti-tumor activity. It inhibits cell proliferation in glioma cells, inducing cell death by apoptosis and/or necroptosis. In the present study, we found that iPA inhibits aerobic glycolysis in unmodified U87MG cells and in the same cell line engineered to over-express wild-type epidermal growth factor receptor (EGFR) or EGFR variant III (vIII), as well as in a primary GBM4 patient-derived cell line. The detection of glycolysis showed that iPA treatment suppressed ATP and lactate production. We also evaluated the response of iPA treatment in normal human astrocyte primary cells, healthy counterpart cells of the brain. Aerobic glycolysis in treated normal human astrocyte cells did not show significant changes compared to GBM cells. To determine the mechanism of iPA action on aerobic glycolysis, we investigated the expression of certain enzymes involved in this metabolic pathway. We observed that iPA reduced the expression of pyruvate kinase M2 (PKM2), which plays a key role in the regulation of aerobic glycolysis, promoting tumor cell proliferation. The reduction of PKM2 expression is a result of the inhibition of the inhibitor of nuclear factor kappa-B kinase subunit, beta/nuclear factor-kappa B pathway upon iPA treatment. In conclusion, these experimental results show that iPA may inhibit aerobic glycolysis of GBM in stabilized cell lines and primary GBM cells by targeting the expression and activity of PKM2.

N6-Isopentenyladenosine Hinders the Vasculogenic Mimicry in Human Glioblastoma Cells through Src-120 Catenin Pathway Modulation and RhoA Activity Inhibition.

BACKGROUND: Vasculogenic mimicry (VM) is a functional microcirculation pattern formed by aggressive tumor cells. Thus far, no effective drugs have been developed to target VM. Glioblastoma (GBM) is the most malignant form of brain cancer and is a highly vascularized tumor. Vasculogenic mimicry represents a means whereby GBM can escape anti-angiogenic therapies. METHODS: Here, using an in vitro tube formation assay on Matrigel, we evaluated the ability of N6-isopentenyladenosine (iPA) to interfere with vasculogenic mimicry (VM). RhoA activity was assessed using a pull-down assay, while the modulation of the adherens junctions proteins was analyzed by Western blot analysis. RESULTS: We found that iPA at sublethal doses inhibited the formation of capillary-like structures suppressing cell migration and invasion of U87MG, U343MG, and U251MG cells, of patient-derived human GBM cells and GBM stem cells. iPA reduces the vascular endothelial cadherin (VE-cadherin) expression levels in a dose-dependent manner, impairs the vasculogenic mimicry network by modulation of the Src/p120-catenin pathway and inhibition of RhoA-GTPase activity. CONCLUSIONS: Taken together, our results revealed iPA as a promising novel anti-VM drug in GBM clinical therapeutics.

A highly efficient organogenesis protocol based on zeatin riboside for in vitro regeneration of eggplant.

BACKGROUND: Efficient organogenesis induction in eggplant (Solanum melongena L.) is required for multiple in vitro culture applications. In this work, we aimed at developing a universal protocol for efficient in vitro regeneration of eggplant mainly based on the use of zeatin riboside (ZR). We evaluated the effect of seven combinations of ZR with indoleacetic acid (IAA) for organogenic regeneration in five genetically diverse S. melongena and one S. insanum L. accessions using two photoperiod conditions. In addition, the effect of six different concentrations of indolebutyric acid (IBA) in order to promote rooting was assessed to facilitate subsequent acclimatization of plants. The ploidy level of regenerated plants was studied. RESULTS: In a first experiment with accessions MEL1 and MEL3, significant (p < 0.05) differences were observed for the four factors evaluated for organogenesis from cotyledon, hypocotyl and leaf explants, with the best results obtained (9 and 11 shoots for MEL1 and MEL3, respectively) using cotyledon tissue, 16 h light / 8 h dark photoperiod conditions, and medium E6 (2 mg/L of ZR and 0 mg/L of IAA). The best combination of conditions was tested in the other four accessions and confirmed its high regeneration efficiency per explant when using both cotyledon and hypocotyl tissues. The best rooting media was R2 (1 mg/L IBA). The analysis of ploidy level revealed that between 25 and 50% of the regenerated plantlets were tetraploid. CONCLUSIONS: An efficient protocol for organogenesis of both cultivated and wild accessions of eggplant, based on the use of ZR, is proposed. The universal protocol developed may be useful for fostering in vitro culture applications in eggplant requiring regeneration of plants and, in addition, allows developing tetraploid plants without the need of antimitotic chemicals.

Increased endogenous indole-3-acetic acid:abscisic acid ratio is a reliable marker of Pinus massoniana rejuvenation.

Pinus massoniana is a recalcitrant tree species for rooting in vitro. We rejuvenated 26-year-old P. massoniana trees by successive grafting. Rooting rates of rejuvenated shoots were > 83.1% after rooting induction. We compared endogenous levels of indole-3-acetic acid (IAA), abscisic acid (ABA), gibberellins (GAs) and zeatin-riboside (ZR), and the rhizogenesis ability of axillary shoots of mature and rejuvenated materials in vitro, i.e., somaplants and grafts. Enhancement of the rooting ability of mature materials in vitro following somatic embryogenesis or repeated grafting onto juvenile rootstocks was accompanied by increased IAA and GAs levels, and by decreased ABA levels in scions used as starting material for micropropagation in vitro. Successive subcultures did not influence the rooting ability of shoots from untreated mature material. Rooting ability of shoots in vitro, however, gradually increased with subculture frequency during repeated subculturing in grafting materials. The IAA:ABA ratio in shoots in vitro after grafting five times, and consequently capable of root organogenesis, was higher than in shoots of untreated mature material incapable of root organogenesis in vitro. A high IAA:ABA ratio was detected in scions of somaplants that were capable of rooting in vitro despite subculture times. We found that the endogenous IAA:ABA ratio is a reliable marker for the recovery of root organogenesis in vitro after rejuvenating treatments for mature P. massoniana trees.

N6-isopentenyladenosine a new potential anti-angiogenic compound that targets human microvascular endothelial cells in vitro.

N6-isopentenyladenosine is an anti-proliferative and pro-apoptotic atypical nucleoside for normal and tumor cells. Considering the role of angiogenesis in various diseases, we investigated the cytotoxic effect of N6-isopentenyladenosine on human microvascular endothelial cells, protagonists in angiogenesis. Our results show that N6-isopentenyladenosine induced a significant reduction of cell viability, upregulated p21 and promoted caspase-3 cleavage in a dose dependent manner leading to apoptotic cell death as detected by FACS analysis. To understand structure-function relationship of N6-isopentenyladenosine, we investigated the effect of some N6-isopentenyladenosine analogs. Our results suggest that N6-isopentenyladenosine and some of its derivatives are potentially novel angiostatic agents and might be associated with other anti-angiogenic compounds for a better outcome.

Design, synthesis and perception of fluorescently labeled isoprenoid cytokinins.

Isoprenoid cytokinins play a number of crucial roles in the regulation of plant growth and development. To study cytokinin receptor properties in plants, we designed and prepared fluorescent derivatives of 6-[(3-methylbut-2-en-1-yl)amino]purine (N6-isopentenyladenine, iP) with several fluorescent labels attached to the C2 or N9 atom of the purine moiety via a 2- or 6-carbon linker. The fluorescent labels included dansyl (DS), fluorescein (FC), 7-nitrobenzofurazan (NBD), rhodamine B (RhoB), coumarin (Cou), 7-(diethylamino)coumarin (DEAC) and cyanine 5 dye (Cy5). All prepared compounds were screened for affinity for the Arabidopsis thaliana cytokinin receptor (CRE1/AHK4). Although the attachment of the fluorescent labels to iP via the linkers mostly disrupted binding to the receptor, several fluorescent derivatives interacted well. For this reason, three derivatives, two rhodamine B and one 4-chloro-7-nitrobenzofurazan labeled iP were tested for their interaction with CRE1/AHK4 and Zea mays cytokinin receptors in detail. We further showed that the three derivatives were able to activate transcription of cytokinin response regulator ARR5 in Arabidopsis seedlings. The activity of fluorescently labeled cytokinins was compared with corresponding 6-dimethylaminopurine fluorescently labeled negative controls. Selected rhodamine B C2-labeled compounds 17, 18 and 4-chloro-7-nitrobenzofurazan N9-labeled compound 28 and their respective negative controls (19, 20 and 29, respectively) were used for in planta staining experiments in Arabidopsis thaliana cell suspension culture using live cell confocal microscopy.

Recognition by natural killer cells of N6-isopentenyladenosine-treated human glioma cell lines.

Cancer cell stress induced by cytotoxic agents promotes antitumor immune response. Here, we observed that N6-isopentenyladenosine (iPA), an isoprenoid modified adenosine with a well established anticancer activity, was able to induce a significant upregulation of cell surface expression of natural killer (NK) cell activating receptor NK Group 2 member D (NKG2D) ligands on glioma cells in vitro and xenografted in vivo. Specifically suboptimal doses of iPA (0.1 and 1 µM) control the selective upregulation of UL16-binding protein 2 on p53wt-expressing U343MG and that of MICA/B on p53mut-expressing U251MG cells. This event made the glioblastoma cells a potent target for NK cell-mediated recognition through a NKG2D restricted mechanism. p53 siRNA-mediated knock-down and pharmacological inhibition (pifithrin-α), profoundly prevented the iPA action in restoring the immunogenicity of U343MG cells through a mechanism that is dependent upon p53 status of malignancy. Furthermore, accordingly to the preferential recognition of senescent cells by NK cells, we found that iPA treatment was critical for glioma cells entry in premature senescence through the induction of S and G2/M phase arrest. Collectively, our results indicate that behind the well established cytotoxic and antiangiogenic effects, iPA can also display an immune-mediated antitumor activity. The indirect engagement of the innate immune system and its additional activity in primary derived patient's glioma cell model (GBM17 and GBM37), fully increase its translational relevance and led to the exploitation of the isoprenoid pathway for a valid therapeutic intervention in antiglioma research.

The isoprenoid end product N6-isopentenyladenosine reduces inflammatory response through the inhibition of the NFκB and STAT3 pathways in cystic fibrosis cells.

OBJECTIVE: N6-isopentenyladenosine (iPA) is an intermediate of the mevalonate pathway that exhibits various anti-cancer effects. However, studies on its anti-inflammatory activity are scarce and underlying molecular mechanisms are unknown. Therefore, we aimed to investigate the ability of iPA to exert anti-inflammatory effects in the human cystic fibrosis (CF) cell model of exacerbated inflammation. MATERIALS AND METHODS: TNFα-stimulated CF cells CuFi-1 and its normal counterpart NuLi-1 were pre-treated with increasing concentrations of iPA and cell viability and proliferation were assessed by MTT and BrdU assays. The effect of iPA on IL-8 and RANTES secretion was determined by ELISA, and the activation and expression of signaling molecules and selenoproteins were studied by Western blot. To assess the direct effect of iPA on NFκB activity, luciferase assay was performed on TNFα-stimulated HEK293/T cells transfected with a NFκB reporter plasmid. RESULTS: We demonstrated for the first time that iPA prevents IL-8 and RANTES release in TNFα-stimulated CF cells and this effect is mediated by increasing the expression of the direct NFκB inhibitor IκBα and decreasing the levels of STAT3. Consistent with this, we showed that iPA inhibited TNFα-mediated NFκB activation in HEK/293T cells. Finally, we also found that iPA improved the levels of glutathione peroxidase 1 and thioredoxin reductase 1 only in CF cells suggesting its ability to maintain sufficient expression of these anti-oxidant selenoproteins. CONCLUSIONS: Our findings indicate that iPA can exert anti-inflammatory activity especially in the cases of excessive inflammatory response as in CF.

N6-isopentenyladenosine dual targeting of AMPK and Rab7 prenylation inhibits melanoma growth through the impairment of autophagic flux.

Targeting the autophagic process is considered a promising therapeutic strategy in cancer since a great number of tumors, including melanoma, show high basal levels of protective autophagy that contributes to tumor progression and chemoresistance. Here, exploiting both in vitro and in vivo approaches, we identified N6-isopentenyladenosine (iPA), an end product of the mevalonate pathway, as a novel autophagy inhibitor with an interesting anti-melanoma activity. iPA, after being phosphorylated by adenosine kinase into 5'-iPA-monophosphate, induces autophagosome accumulation through AMPK activation, measured by increased fluorescent GFP-LC3 puncta and enhanced conversion into the lipidated autophagosome-associated LC3-II. However, at a later stage iPA blocks the autophagic flux monitored by p62 accumulation, Luciferase reporter-based assay for LC3 turnover in living cells and fluorescence of a tandem RFP-GFP-LC3 construct. Impaired autophagic flux is due to the block of autophagosome-lysosome fusion through the defective localization and function of Rab7, whose prenylation is inhibited by iPA, resulting in a net inhibition of autophagy completion that finally leads to melanoma apoptotic cell death. AMPK silencing prevents apoptosis upon iPA treatment, whereas basal autophagosome turnover is still inhibited due to unprenylated Rab7. These results strongly support the advantage of targeting autophagy for therapeutic gain in melanoma and provide the preclinical rational to further investigate the antitumor action of iPA, able to coordinately induce autophagosome accumulation and inhibit the autophagic flux, independently targeting AMPK and Rab7 prenylation. This property may be particularly useful for the selective killing of tumors, like melanoma, that frequently develop chemotherapy resistance due to protective autophagy activation.

Regulation of maize kernel weight and carbohydrate metabolism by abscisic acid applied at the early and middle post-pollination stages in vitro.

Abscisic acid (ABA) accumulates in plants under drought stress, but views on the role of ABA in kernel formation and abortion are not unified. The response of the developing maize kernel to exogenous ABA was investigated by excising kernels from cob sections at four days after pollination and culturing in vitro with different concentrations of ABA (0, 5, 10, 100µM). When ABA was applied at the early post-pollination stage (EPPS), significant weight loss was observed at high ABA concentration (100µM), which could be attributed to jointly affected sink capacity and activity. Endosperm cells and starch granules were decreased significantly with high concentration, and ABA inhibited the activities of soluble acid invertase and acid cell wall invertase, together with earlier attainment of peak values. When ABA was applied at the middle post-pollination stage (MPPS), kernel weight was observably reduced with high concentration and mildly increased with low concentration, which was regulated due to sink activity. The inhibitory effect of high concentration and the mild stimulatory effect of low concentration on sucrose synthase and starch synthase activities were noted, but a peak level of ADP-glucose pyrophosphorylase (AGPase) was stimulated in all ABA treatments. Interestingly, AGPase peak values were advanced by low concentration and postponed by high concentration. In addition, compared with the control, the weight of low ABA concentration treatments were not statistically significant at the two stages, whereas weight loss from high concentration applied at EPPS was considerably obvious compared with that of the MPPS, but neither led to kernel abortion. The temporal- and dose-dependent impacts of ABA reveal a complex process of maize kernel growth and development.

N6-Isopentenyladenosine promoted HeLa cell apoptosis through inhibitions of AKT and transforming growth factor ß-activated kinase 1 activation.

N6-Isopentenyladenosine, a member of the family of plant hormones, possesses anti-cancer activities on a number of cancer cell lines. However, its mode of action in cervical cancer cell remains poorly understood. Our computational docking studies showed that N6-Isopentenyladenosine could bind with the really interesting new gene domain of tumor necrosis factor receptor-associated factor 6, which is an ubiquitination E3 ligase. Tumor necrosis factor receptor-associated factor 6-mediated ubiquitination is known to activate both protein kinase B (also known as AKT) and transforming growth factor ß-activated kinase 1, and the really interesting new gene domain comprises the core of the ubiquitin ligase catalytic domain. First, we evaluated the effects of iPA on cervical cancer cell line HeLa using MTT and flow cytometry. Second, we examined the effects of iPA on activation of tumor necrosis factor receptor-associated factor 6-mediated downstream targets using western blot or immunoprecipitation. iPA could reduce HeLa cell proliferation through apoptosis, and such anti-cancer activity is associated with inhibitions of both AKT and transforming growth factor ß-activated kinase 1 signaling pathways. In addition, suppression of the anti-apoptotic protein Bcl-2 and elevation of the pro-apoptotic protein Bax were also observed. Anti-proliferation properties of iPA are likely due to its binding at the really interesting new gene domain of tumor necrosis factor receptor-associated factor 6 and loss of AKT and transforming growth factor ß-activated kinase 1 activities as a result of functional modulations of tumor necrosis factor receptor-associated factor 6. These results support the emerging notion that tumor necrosis factor receptor-associated factor 6 could serve as a viable target for developing new cancer therapeutics.

The natural cytokinin 2OH3MeOBAR induces cell death by a mechanism that is different from that of the "classical" cytokinin ribosides.

Cytokinin ribosides (N6-substituted adenosines) have demonstrated anticancer activity in various cultured cell lines, several xenografts and even a small clinical trial. Effects of kinetin riboside, N6-benzyladenosine (BAR) and N6-isopentenyladenosine on various parameters related to apoptosis have also been reported, but not directly compared with those of the highly active naturally occurring aromatic cytokinins oTR (ortho-topolin riboside) and 2OH3MeOBAR (N6-(2-hydroxy-3-methoxybenzyl)adenosine). Here we show that 2OH3MeOBAR is the most active cytokinin riboside studied to date (median, 1st quartile, 3rd quartile and range of GI50 in tests with the NCI60 cell panel: 0.19, 0.10, 0.43 and 0.02 to 15.7 µM, respectively) and it differs from other cytokinins by inducing cell death without causing pronounced ATP depletion. Analysis of NCI60 test data suggests that its activity is independent of p53 status. Further we demonstrate that its 5'-monophosphate, the dominant cancer cell metabolite, inhibits the candidate oncogene DNPH1. Synthesis, purification, HPLC-MS identification and HPLC-UV quantification of 2OH3MeOBAR metabolites are also reported.

Antiglioma effects of N6-isopentenyladenosine, an endogenous isoprenoid end product, through the downregulation of epidermal growth factor receptor.

Malignant gliomas are highly dependent on the isoprenoid pathway for the synthesis of lipid moieties critical for cell proliferation. The isoprenoid derivative N6-isopentenyladenosine (iPA) displays pleiotropic biological effects, including a direct anti-tumor activity in several tumor models. The antiglioma effects of iPA was then explored in U87MG cells both in vitro and grafted in mice and the related molecular mechanism confirmed in primary derived patients' glioma cells. iPA powerfully inhibited tumor cell growth and induced caspase-dependent apoptosis through a mechanism involving a marked accumulation of the pro-apoptotic BIM protein and inhibition of EGFR. Indeed, activating AMPK following conversion into its iPAMP active form, iPA stimulated EGFR phosphorylation and ubiquitination along a proteasome-mediated pathway which was responsible for receptor degradation and its downstream signaling pathways inhibition, including the STAT3, ERK and AKT cascade. The inhibition of AMPK by compound C prevented iPA-mediated phosphorylation of EGFR, known to precede receptor loss. As expected the block of EGFR degradation, by exposure to the proteasome inhibitor MG132, significantly reduced iPA-induced cell death. Given the importance of receptor degradation in iPA-mediated cytotoxicity, we also documented that the EGFR expression levels in a panel of primary glioma cells confers them a high sensitivity to iPA treatment. In conclusion our study provides the first evidence of iPA antiglioma effect. Indeed, as glioma is driven by aberrant signaling of growth factor receptors, particularly the EGFR, iPA, alone or in association with EGFR targeted therapies, might be a promising therapeutic tool to achieve a potent anti-tumoral effect.

Biological Activity of Vegetal Extracts Containing Phenols on Plant Metabolism.

The influence of vegetal extracts derived from red grape, blueberry fruits and hawthorn leaves on Zea mays L. plant growth and the activity of phenylalanine ammonia-lyase (PAL), a key enzyme of the phenylpropanoid pathway, was investigated in laboratory experiments. The extracts were characterized using FT-IR and Raman spectroscopies in order to obtain a pattern of the main functional groups. In addition, phenols content was determined by HPLC, whereas the content of indoleacetic acid and isopentenyladenosine hormones was determined by ELISA test and the auxin and gibberellin-like activities by plant-bioassays. The treated maize revealed increased root and leaf biomass, chlorophyll and sugars content with respect to untreated plants. Hawthorn, red grape skin and blueberry at 1.0 mL/L induced high p-coumaric content values, whilst hawthorn also showed high amounts of gallic and p-hydroxybenzoic acids. PAL activity induced by hawthorn at 1.0 mL/L had the highest values (11.1-fold UNT) and was strongly and linearly related with the sum of leaf phenols. Our results suggest that these vegetal extracts contain more than one group of plant-promoting substances.

Chemical modification of the plant isoprenoid cytokinin N(6)-isopentenyladenosine yields a selective inhibitor of human enterovirus 71 replication.

In this study, we demonstrate that N(6)-isopentenyladenosine, which essentially is a plant cytokinin-like compound, exerts a potent and selective antiviral effect on the replication of human enterovirus 71 with an EC50 of 1.0 ± 0.2 µM and a selectivity index (SI) of 5.7. The synthesis of analogs with modification of the N(6)-position did not result in a lower EC50 value. However, in particular with the synthesis of N(6)-(5-hexene-2-yne-1-yl)adenosine (EC50 = 4.3 ± 1.5 µM), the selectivity index was significantly increased: because of a reduction in the adverse effect of this compound on the host cells, an SI > 101 could be calculated. With this study, we for the first time provide proof that a compound class that is based on the plant cytokinin skeleton offers an interesting starting point for the development of novel antivirals against mammalian viruses, in the present context in particular against enterovirus 71.

The plant hormone zeatin riboside inhibits T lymphocyte activity via adenosine A2A receptor activation.

Cytokinins are plant hormones that play an integral role in multiple aspects of plant growth and development. The biological functions of cytokinins in mammalian systems are, however, largely uncharacterized. The naturally occurring cytokinin zeatin riboside has recently been demonstrated to activate the mammalian adenosine A(2A) receptor, which is broadly expressed by various cell types including immune system cells, with the activation of the A(2A)R playing a role in the regulation of cells involved in both innate and adaptive immunity. We show for the first time that zeatin riboside modulates mammalian immune system activity via an A(2A)R-dependent mechanism. Specifically, zeatin riboside treatment induces the production of cyclic adenosine monophosphate (cAMP) by T lymphocytes and inhibits the production by CD3(+)CD4(+) T cells of interferon (IFN)-γ, IL-2, tumor-necrosis factor (TNF)-α, IL-4 and IL-13, and the production by CD3(+)CD8(+) T cells of IFN-γ, IL-2 and TNF-α. Additionally, the upregulation of CD25, CD69 and CD40L by activated T lymphocytes is modulated by zeatin riboside. Zeatin riboside treatment also potently inhibits thioglycollate-induced peritoneal leukocytosis. The immunomodulatory activities of zeatin riboside are blocked by co-treatment with the selective A(2A)R antagonist ZM241385. These data suggest that zeatin riboside possesses therapeutic potential as a mammalian immunomodulatory agent.

Plant regeneration and biochemical accumulation of hydroxybenzoic and hydroxycinnamic acid derivatives in Hypoxis hemerocallidea organ and callus cultures.

Micropropagation of Hypoxis hemerocallidea Fisch. and C.A. Mey was used as a model system to study the influence of cytokinins (CKs) on plant regeneration and biochemical accumulation of hydroxybenzoic and hydroxycinnamic acid derivatives in organ and callus cultures and their antioxidant activity. Fourteen free phenolic acids were detected using ultra performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) while antioxidant activity was evaluated using oxygen radical absorbance capacity (ORAC) and 2,2-diphenyl-1-picryl hydrazyl (DPPH) radical scavenging activity. Cytokinins had a significant effect on the biochemical accumulation of hydroxybenzoic and hydroxycinnamic acid derivatives in H. hemerocallidea organ cultures. In particular, meta-topolin-treated organ cultures produced high concentrations of gallic, protocatechuic, gentisic, p-hydroxybenzoic, m-hydroxybenzoic, salicylic, chlorogenic and trans-cinnamic acids. The isoprenoid CK, N(6)-(2-isopentenyl)-adenine significantly increased the accumulation of hydroxycinnamic acid derivatives, namely, caffeic, p-coumaric, sinapic and ferulic acids. Cytokinin-treated organ cultures exhibited a significant increase in antioxidant activity, particularly in the ORAC model. In callus cultures, CKs decreased the concentrations of hydroxycinnamic acid derivatives and antioxidant activity when compared to the control. Overall, both CK type and concentration had a significant effect on plant regeneration, callus proliferation, biochemical accumulation of free phenolic acids and antioxidant activity of the resultant extracts.

N6-isopentenyladenosine affects cytotoxic activity and cytokines production by IL-2 activated NK cells and exerts topical anti-inflammatory activity in mice.

N6-isopentenyladenosine (iPA) is a modified adenosine with an isopentenyl moiety derived from the mevalonate pathway which displays pleiotropic biological effects, including anti-tumor and anti-angiogenic activity. Previous evidence revealed a biphasic effect of iPA on phytohemagglutinin-stimulated lymphocytes, being pro-proliferative at low doses and anti-proliferative at high doses. Analogously, we have recently shown that low iPA concentrations (<1µM) increased the immune response of natural killer (NK) cells against cancer targets. In the present study, we evaluated the effect of iPA at high concentration (10µM) on IL-2-activated NK cells. iPA, inhibited NK cell proliferation and cytotoxicity against their conventional tumor target, human K562 cells. This inhibition was associated with decreased expression and functionality of NK cell activating receptors NKp44 and NKG2D as well as impaired cyto/chemokines secretion (RANTES, MIP-1α, TNF-α and IFN-γ). ERK/MAPK and STAT5 activation in IL-2-activated NK cells were inhibited by iPA. The results obtained in vitro were validated in vivo in the inflammatory murine model of croton oil-induced ear dermatitis. The topical application of iPA significantly reduced mouse ear oedema, thus suggesting anti-inflammatory properties of this molecule. These results show the ability of iPA to exert anti-inflammatory effects both in vitro and in vivo directly targeting NK cells, providing a novel pharmacological tool in those diseases characterized by a deregulated immune-response, such as cancer or inflammatory conditions.

Effect of shooting medium and source of material on grapevine (Vitis vinifera L.) shoot tip recovery after cryopreservation.

BACKGROUND: Selecting experimental material at the optimal physiological stage is of paramount importance for successful cryopreservation. OBJECTIVE: The study was to investigate the effect of the physiological state of grapevine buds on their regrowth after liquid nitrogen exposure. METHODS: In a first set of experiments, we tested the regrowth of cryopreserved buds sampled from microcuttings cultured on shooting medium containing benzylaminopurine or zeatin riboside for various durations. In a second set of experiments, we studied the regrowth after liquid nitrogen exposure of buds sampled from different positions on the stem of in vitro plantlets. RESULTS: Regrowth of cryopreserved buds sampled from microcuttings was higher (30%), compared to buds sampled directly from in vitro plantlets (23%), for all culture durations of microcuttings on shooting medium tested (2-6 weeks). Addition of cytokinin in the shooting medium improved regrowth of cryopreserved buds compared to buds sampled from microcuttings cultured on medium devoid of growth regulators; however similar results were obtained with the two cytokinins tested. Buds sampled on nodes 3-4 and 6-7 (from the top of the stem) displayed higher regrowth compared to shoot tips. No significant differences were noted in regrowth after cryopreservation between buds sampled from microcuttings produced from the terminal node, or nodes 3-4 and 6-7. CONCLUSION: The physiological state of the plant material is important for cryopreservation success. Actively growing buds sampled from microcuttings displayed higher regrowth compared to buds sampled directly on in vitro plantlets.

N(6)-isopentenyladenosine and analogs activate the NRF2-mediated antioxidant response.

N(6)-isopentenyladenosine (i(6)A), a naturally occurring modified nucleoside, inhibits the proliferation of human tumor cell lines in vitro, but its mechanism of action remains unclear. Treatment of MCF7 human breast adenocarcinoma cells with i(6)A or with three synthetic analogs (allyl(6)A, benzyl(6)A, and butyl(6)A) inhibited growth and altered gene expression. About 60% of the genes that were differentially expressed in response to i(6)A treatment were also modulated by the analogs, and pathway enrichment analysis identified the NRF2-mediated oxidative stress response as being significantly modulated by all four compounds. Luciferase reporter gene assays in transfected MCF7 cells confirmed that i(6)A activates the transcription factor NRF2. Assays for cellular production of reactive oxygen species indicated that i(6)A and analogs had antioxidant effects, reducing basal levels and inhibiting the H2O2- or 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced production in MCF7 or dHL-60 (HL-60 cells induced to differentiate along the neutrophilic lineage) cell lines, respectively. In vivo, topical application of i(6)A or benzyl(6)A to mouse ears prior to TPA stimulation lessened the inflammatory response and significantly reduced the number of infiltrating neutrophils. These results suggest that i(6)A and analogs trigger a cellular response against oxidative stress and open the possibility of i(6)A and benzyl(6)A being used as topical anti-inflammatory drugs.