Proanthocyanidins from the American Cranberry (Vaccinium macrocarpon) inhibit matrix metalloproteinase‐2 and matrix metalloproteinase‐9 activity in human prostate cancer cells via alterations in multiple cellular signalling pathways

Prostate cancer is one of the most common cancers in the Western world, and it is believed that an individual's diet affects his risk of developing cancer. There has been an interest in examining phytochemicals, the secondary metabolites of plants, in order to determine their potential anti‐cancer activities in vitro and in vivo. In this study we document the effects of proanthocyanidins (PACs) from the American Cranberry (Vaccinium macrocarpon) on matrix metalloproteinase (MMP) activity in DU145 human prostate cancer cells. Cranberry PACs decreased cellular viability of DU145 cells at a concentration of 25 µg/ml by 30% after 6 h of treatment. Treatment of DU145 cells with PACs resulted in an inhibition of both MMPs 2 and 9 activity. PACs increased the expression of TIMP‐2, a known inhibitor of MMP activity, and decreased the expression of EMMPRIN, an inducer of MMP expression. PACs decreased the expression of PI‐3 kinase and AKT proteins, and increased the phosphorylation of both p38 and ERK1/2. Cranberry PACs also decreased the translocation of the NF‐κB p65 protein to the nucleus. Cranberry PACs increased c‐jun and decreased c‐fos protein levels. These results suggest that cranberry PACs decreases MMP activity through the induction and/or inhibition of specific temporal MMP regulators, and by affecting either the phosphorylation status and/or expression of MAP kinase, PI‐3 kinase, NF‐κB and AP‐1 pathway proteins. This study further demonstrates that cranberry PACs are a strong candidate for further research as novel anti‐cancer agents. J. Cell. Biochem. 111: 742–754, 2010. © 2010 Wiley‐Liss, Inc.     

Phagocytosis of autologous lymphocytes by cervical preneoplastic and neoplastic cells

Eighty-nine random Pap smears of the uterine cervix were examined to evaluate the phagocytic abnormal cells (PACs) of atypical, dysplastic and neoplastic tissues against infiltrated blood cells. The results revealed that none of the PACs have been identified in atypical (II) and mild dysplastic cells (IIIa). Low levels (1.2%) of PACs were initially demonstrated in patients with moderate dysplasia (IIIb) that increased 1.7-fold in subsequent severe dysplasia (IIIc) and further increased 2.8-fold in invasive carcinomas of the cervix (V). In addition, the data showed age-related responsiveness toward the development of PACs, where 82% of old patients have developed phagocytic activity in moderate dysplastic cells compared with 27% of young patients. However, the difference became less significant at the subsequent classes of the disease. These data further demonstrated that PACs are class-dependent and it may explain one mechanism by which precancerous cells escape immunosurveillance.     

Skin autofluorescence predicts cardio-renal outcome in type 1 diabetes: a longitudinal study

Type 1 diabetes is associated with increased cardiovascular disease (CVD). Decreased endothelial progenitor cells (EPCs) number plays a pivotal role in reduced endothelial repair and development of CVD. We aimed to determine if cardioprotective effect of metformin is mediated by increasing circulating endothelial progenitor cells (cEPCs), pro-angiogenic cells (PACs) and decreasing circulating endothelial cells (cECs) count whilst maintaining unchanged glycemic control. This study was an open label and parallel standard treatment study. Twenty-three type 1 diabetes patients without overt CVD were treated with metformin for 8 weeks (treatment group-TG). They were matched with nine type 1 diabetes patients on standard treatment (SG) and 23 age- and sex-matched healthy volunteers (HC). Insulin dose was adjusted to keep unchanged glycaemic control. cEPCs and cECs counts were determined by flow cytometry using surface markers CD45dimCD34+VEGFR-2+ and CD45dimCD133−CD34+CD144+ respectively. Peripheral blood mononuclear cells were cultured to assess changes in PACs number, function and colony forming units (CFU-Hill’s colonies). At baseline TG had lower cEPCs, PACs, CFU-Hills’ colonies and PACs adhesion versus HC (p < 0.001-all variables) and higher cECs versus HC (p = 0.03). Metformin improved cEPCs, PACs, CFU-Hill’s colonies number, cECs and PACs adhesion (p < 0.05-all variables) to levels seen in HC whilst HbA1c (one-way ANOVA p = 0.78) and glucose variability (average glucose, blood glucose standard deviation, mean amplitude of glycaemic excursion, continuous overall net glycaemic action and area under curve) remained unchanged. No changes were seen in any variables in SG. There was an inverse correlation between CFU-Hill’s colonies with cECs. Metformin has potential cardio-protective effect through improving cEPCs, CFU-Hill’s colonies, cECs, PACs count and function independently of hypoglycaemic effect. This finding needs to be confirmed by long term cardiovascular outcome studies in type 1 diabetes. Trial registration ISRCTN26092132     

Circadian Modulation of c-Fos Expression Occurs only in the SCN and not in Other Visual Projection Areas in the Rat

Brief photic stimuli at different circadian times induce differential expression of c-Fos in the suprachiasmatic nuclei (SCN). Whether circadian modulation of light-induced c-Fos expression occurs in other visual projection areas is not known. We addressed this question by estimating the immunohistochemical expression of c-Fos induced by 60 min light pulses at three different circadian times. The areas studied were the SCN, the ventral lateral geniculate nucleus, the intergeniculate leaflet, the ventral tegmental area, the superior colliculus and a non-visual control, the paraventricular thalamic nucleus (PVT). Light pulses induced an increase in the number of c-Fos immunoreactive cells in the SCN as a function of the circadian time. Remaining visual structures showed a light-induced increase in c-Fos expression but this was not dependent on the circadian time. The non-visual control area (PVT) did not respond to light pulses. Since no circadian modulation was found in the intergeniculate leaflet, which rec eives collateral projections from the same retinal ganglion cells that project to the SCN, nor in other primary visual projection areas, the present findings suggest that the circadian modulation of light-induced c-Fos expression in the SCN depends mainly on the functional properties of its intrinsic neurons.     

Circadian Modulation of c-Fos Expression Occurs only in the SCN and not in Other Visual Projection Areas in the Rat

Brief photic stimuli at different circadian times induce differential expression of c-Fos in the suprachiasmatic nuclei (SCN). Whether circadian modulation of light-induced c-Fos expression occurs in other visual projection areas is not known. We addressed this question by estimating the immunohistochemical expression of c-Fos induced by 60 min light pulses at three different circadian times. The areas studied were the SCN, the ventral lateral geniculate nucleus, the intergeniculate leaflet, the ventral tegmental area, the superior colliculus and a non-visual control, the paraventricular thalamic nucleus (PVT). Light pulses induced an increase in the number of c-Fos immunoreactive cells in the SCN as a function of the circadian time. Remaining visual structures showed a light-induced increase in c-Fos expression but this was not dependent on the circadian time. The non-visual control area (PVT) did not respond to light pulses. Since no circadian modulation was found in the intergeniculate leaflet, which rec eives collateral projections from the same retinal ganglion cells that project to the SCN, nor in other primary visual projection areas, the present findings suggest that the circadian modulation of light-induced c-Fos expression in the SCN depends mainly on the functional properties of its intrinsic neurons.     

Murine T-cell hybridomas that produce lymphokine with macrophage-activating factor activity as a constitutive product

Responder spleen cells primed to alloantigens in vivo could generate high degree of cytotoxicity against low- or nonimmunogenic stimulators such as thymocytes or uv light-treated spleen cells in vitro. However, a removal of adherent cells from primed responder cells remarkably reduced the cytotoxicity after stimulation with such low-immunogenic stimulators. Adding a small number of peritoneal adherent cells (PACs) also suppressed the cytotoxic activity of unseparated responders against low-immunogenic stimulators. These suppressive effects by PACs were blocked by indomethacin. By adding prostaglandin E₂, cytotoxic T lymphocyte (CTL) generation of primed unseparated responders against low-immunogenic stimulators was suppressed; however, cytotoxic activity against mitomycin C-treated stimulators was not suppressed. These results suggested that prostaglandins released from PACs selectively inhibited the function of splenic adherent cells that were required for CTL generation of primed responder spleen cells against low-immunogenic stimulators in vitro.     

InsP₃receptors and Orai channels in pancreatic acinar cells: co-localization and its consequences

Orai1 proteins have been recently identified as subunits of SOCE (store-operated Ca2? entry) channels. In primary isolated PACs (pancreatic acinar cells), Orai1 showed remarkable co-localization and co-immunoprecipitation with all three subtypes of IP?Rs (InsP? receptors). The co-localization between Orai1 and IP?Rs was restricted to the apical part of PACs. Neither co-localization nor co-immunoprecipitation was affected by Ca2? store depletion. Importantly we also characterized Orai1 in basal and lateral membranes of PACs. The basal and lateral membranes of PACs have been shown previously to accumulate STIM1 (stromal interaction molecule 1) puncta as a result of Ca2? store depletion. We therefore conclude that these polarized secretory cells contain two pools of Orai1: an apical pool that interacts with IP?Rs and a basolateral pool that interacts with STIM1 following the Ca2? store depletion. Experiments on IP?R knockout animals demonstrated that the apical Orai1 localization does not require IP?Rs and that IP?Rs are not necessary for the activation of SOCE. However, the InsP?-releasing secretagogue ACh (acetylcholine) produced a negative modulatory effect on SOCE, suggesting that activated IP?Rs could have an inhibitory effect on this Ca2? entry mechanism.     

A reliable and efficient method for deleting operational sequences in PACs and BACs

P1-derived artificial chromosomes (PACs) and bacterial artificial chromosomes (BACs) have become very useful as tools to study gene expression and regulation in cells and in transgenic mice. They carry large fragments of genomic DNA (≥100 kb) and therefore may contain all of the cis-regulatory elements required for expression of a gene. Because of this, even when inserted randomly in the genome, they can emulate the native environment of a gene resulting in a tightly regulated pattern of expression. Because these large genomic clones often contain DNA sequences which can manipulate chromatin at the local level, they become immune to position effects which affect expression of smaller transgenes, and thus their expression is proportional to copy number. Transgenic mice containing large BACs and PACs have become excellent models to examine the regulation of gene expression. Their usefulness would certainly be increased if easy and efficient methods are developed to manipulate them. We describe herein a method to make deletion mutations reliably and efficiently using a novel modification of the Chi-stimulated homologous recombination method. Specifically, we generated and employed a Lox511 ‘floxed’ CAM resistance marker that first affords selection for homologous recombination in Escherichia coli, and then can be easily deleted leaving only a single Lox511 site as the footprint.     

TRPM4 links calcium signaling to membrane potential in pancreatic acinar cells

Transient receptor potential cation channel subfamily M member 4 (TRPM4) is a Ca²⁺-activated nonselective cation channel that mediates membrane depolarization. Although, a current with the hallmarks of a TRPM4-mediated current has been previously reported in pancreatic acinar cells (PACs), the role of TRPM4 in the regulation of acinar cell function has not yet been explored. In the present study, we identify this TRPM4 current and describe its role in context of Ca²⁺ signaling of PACs using pharmacological tools and TRPM4-deficient mice. We found a significant Ca²⁺-activated cation current in PACs that was sensitive to the TRPM4 inhibitors 9-phenanthrol and 4-chloro-2-[[2-(2-chlorophenoxy)acetyl]amino]benzoic acid (CBA). We demonstrated that the CBA-sensitive current was responsible for a Ca²⁺-dependent depolarization of PACs from a resting membrane potential of −44.4 ± 2.9 to −27.7 ± 3 mV. Furthermore, we showed that Ca²⁺ influx was higher in the TRPM4 KO- and CBA-treated PACs than in control cells. As hormone-induced repetitive Ca²⁺ transients partially rely on Ca²⁺ influx in PACs, the role of TRPM4 was also assessed on Ca²⁺ oscillations elicited by physiologically relevant concentrations of the cholecystokinin analog cerulein. These data show that the amplitude of Ca²⁺ signals was significantly higher in TRPM4 KO than in control PACs. Our results suggest that PACs are depolarized by TRPM4 currents to an extent that results in a significant reduction of the inward driving force for Ca²⁺. In conclusion, TRPM4 links intracellular Ca²⁺ signaling to membrane potential as a negative feedback regulator of Ca²⁺ entry in PACs.     

Acid sphingomyelinase is indispensable for UV light-induced Bax conformational change at the mitochondrial membrane

Ultraviolet light-induced apoptosis can be caused by DNA damage but also involves immediate-early cell death cascades characteristic of death receptor signaling. Here we show that the UV light-induced apoptotic signaling pathway is unique, targeting Bax activation at the mitochondrial membrane independent of caspase-8 or cathepsin D activity. Cells deficient in acid sphingomyelinase (ASMase) do not show UV light-induced Bax activation, cytochrome c release, or apoptosis. In ASMase-deficient cells, the apoptotic UV light response is restored by stable or transient expression of human ASMase. Bax conformational change in ASMase(-/-) cells is also caused by synthetic C(16)-ceramide acting on intact cells or isolated mitochondria. The results suggest that UV light-triggered ASMase activation is essentially required for Bax conformational change leading to mitochondrial release of pro-apoptotic factors like cytochrome c and Smac.     

Proanthocyanidins alleviate pentylenetetrazole-induced epileptic seizures in mice via the antioxidant activity

The role of oxidative stress in the initiation and progress of epilepsy is well established. Proanthocyanidins (PACs), a naturally occurring polyphenolic compound, have been reported to possess a broad spectrum of pharmacological and therapeutic properties against oxidative stress. However, the protective effects of proanthocyanidins against epilepsy have not been clarified. In the present study, we used the pentylenetetrazole (PTZ)-induced epilepsy mouse model to explore whether proanthocyanidins could help to reduce oxidative stress and protect against epilepsy. Mice were allocated into four groups (n?=?14 per each group): control, PTZ (60 mg/kg, intraperitoneally), PACs?+?PTZ (200 mg/kg, p.o.) and sodium valproate (VPA)?+?PTZ (200 mg/kg, p.o.). PTZ injection caused oxidative stress in the hippocampal tissue as represented by the elevated lipid peroxidation and NO synthesis and increased expression of iNOS. Furthermore, depleted levels of anti-oxidants, GSH, GR, GPx, SOD, and CAT also indicate that oxidative stress was induced in mice exposed to PTZ. Additionally, a state of neuroinflammation was recorded following the developed seizures. Moreover, neuronal apoptosis was recorded following the development of epileptic convulsions as confirmed by the elevated Bax and caspase-3 and the decreased Bcl2 protein. Moreover, AChE activity, DA, NE, 5-HT, brain-derived neurotrophic factor levels, and gene expression of Nrf2 have decreased in the hippocampal tissue of PTZ exposed mice. However, pre-treatment of mice with PACs protected against the generation of oxidative stress, apoptosis, and neuroinflammation in the PTZ exposed mice brain as the biomarkers for all these conditions was bought to control levels. In addition, the gene expression of Nrf2 was significantly upregulated following PACs treatment. These results suggest that PACs can ameliorate oxidative stress, neuroinflammation, and neuronal apoptosis by activating the Nrf2 signaling pathway in PTZ induced seizures in mice.

     

Effect of phytochemical concentrations on biological activities of cranberry extracts

Plants of cranberry (Vaccinium macrocarpon) furnish edible fruits and derivates that have been used for the prevention and treatment of urinary tract infections. In the present work we compare two commercial extracts that contain proanthocyanins (PACs) at 4 percent and 20 percent for antimicrobial, antiproliferative, antiradical and protective properties against oxidative stress on cell lines. Both extracts showed antimicrobial activity (MIC values range 3-100 microg/ml). Extract at 20 percent PACs showed higher antiproliferative activity against HepG2 and MCF7 cells, but not against C2C12 cells. Both extracts showed a dose-dependent free-radical scavenging capacity, and a protective effect on the cell damage was also revealed by reduction of intracellular active oxygen species release. Cranberry extracts confirmed antioxidative properties and efficacy in reduction of cell viability that resulted stronger against tumor cells. The pretreatment with cranberry extracts, furthermore, reveal an increase of cell resistance against oxidative stress, suggesting a potential role as a dietary supplement in preventing free-radical damage. The proanthocyanidin content is critical to determine the extract efficacy. In cellular experiments the extracts resulted clearly differentiated in their activity, and the activity was strongly influenced by PACs content. Only in DPPH test the free radical scavenging activity seemed to be directly related to proanthocyanidins content.     

Activity and characterization of mixed organic compounds extracted from Rhodobacter sphaeroides as alternative materials to serum for mammalian cell growth

Photosynthetic microorganisms produce relatively large amounts of physiologically active materials which stimulate the physiological activity of other organisms. In this study, mammalian HeLa cells were cultured in different culture media which were Dulbecco's modified Eagle medium (DMEM) with newborn calf serum (NCS), and DMEM including different types of physiologically activating compounds (PACs) extracted from Rhodobacter sphaeroides grown under various culture conditions. R. sphaeroides was grown under the following five different culture conditions: anaerobically in the light, anaerobically in the dark and treated with dimethyl sulfoxide, aerobically in the dark for 48 h, in the light for 48 h, and in the light for 24 h and changed after previous culturing in the dark for 24 h. The growth of HeLa cell was measured by cell counting using a hemocytometer, and the fluorescent intensities of cellular lysosomes were measured to check the level of cellular stress caused by adding PACs. The growth of HeLa cells cultured in DMEM with PACs extracted from R. sphaeroides aerobically grown under dark conditions was enhanced compared to that of cells grown with NCS. We also found that a high concentration of pigments such as bacteriochlorophylls and carotenoids and a high concentration of arginine produced by R. sphaeroides aerobically grown in the dark were implicated in increased growth of the HeLa cells. Therefore, our results suggest that PACs extracted from R. sphaeroides aerobically cultured in dark conditions can enhance the physiological activity of mammalian cells and serve as nontoxic and bioavailable materials.     

Polyamine oxidase,a hydrogen peroxide-producing enzyme,is up-regulated by light and down-regulated by auxin in the outer tissues of the maize mesocotyl

Exogenously supplied auxin (1-naphthaleneacetic acid) inhibited light-induced activity increase of polyamine oxidase (PAO), a hydrogen peroxide-producing enzyme, in the outer tissues of maize (Zea mays) mesocotyl. The same phenomenon operates at PAO protein and mRNA accumulation levels. The wall-bound to extractable PAO activity ratio was unaffected by auxin treatment, either in the dark or after light exposure. Ethylene treatment did not affect PAO activity, thus excluding an effect of auxin via increased ethylene biosynthesis. The auxin polar transport inhibitors N(1)-naphthylphthalamic acid or 2,3,5-triiodobenzoic acid caused a further increase of PAO expression in outer tissues after light treatment. The small increase of PAO expression, normally occurring in the mesocotyl epidermis during plant development in the dark, was also inhibited by auxin, although to a lesser extent with respect to light-exposed tissue, and was stimulated by N(1)-naphthylphthalamic acid or 2,3,5-triiodobenzoic acid, thus suggesting a complex regulation of PAO expression. Immunogold ultrastructural analysis in epidermal cells revealed the association of PAO with the secretory pathway and the cell walls. The presence of the enzyme in the cell walls of this tissue greatly increased in response to light treatment. Consistent with auxin effects on light-induced PAO expression, the hormone treatment inhibited the increase in immunogold staining both intraprotoplasmically and in the cell wall. These results suggest that both light and auxin finely tune PAO expression during the light-induced differentiation of the cell wall in the maize mesocotyl epidermal tissues.     

Phospholipase A2beta mediates light-induced stomatal opening in Arabidopsis

Phospholipase A(2) (PLA(2)) catalyses the hydrolysis of phospholipids into lysophospholipids and free fatty acids. Physiological studies have indicated that PLA(2) is involved in stomatal movement. However, genetic evidence of a role of PLA(2) in guard cell signalling has not yet been reported. To identify PLA(2) gene(s) that is (are) involved in light-induced stomatal opening, stomatal movement was examined in Arabidopsis thaliana plants in which the expression of PLA(2) isoforms was reduced or knocked-out. Light-induced stomatal opening in PLA(2)alpha knockout plants did not differ from wild-type plants. Plants in which PLA(2)beta was silenced by RNA interference exhibited delayed light-induced stomatal opening, and this phenotype was reversed by exogenous lysophospholipids, which are products of PLA(2). Stomatal opening in transgenic plants that over-expressed PLA(2)beta was faster than wild-type plants. The expression of PLA(2)beta was localized to the endoplasmic reticulum of guard cells, and increased in response to light in the mature leaf. Aristolochic acid, which inhibits light-induced stomatal opening, inhibited the activity of purified PLA(2)beta. Collectively, these results provide evidence that PLA(2)beta is involved in light-induced stomatal opening in Arabidopsis.     

Effect of charge redistribution on the binding of DNA nucleotide to carbon nanotube in molecular dynamics

All-atom molecular dynamics (MD) simulations are performed to study the binding of DNA nucleotides with two carbon nanotubes (CNTs) with similar diameters but different chiralities. Two schemes for assigning partial atomic charges (PACs) are adopted: (I) using PACs obtained from isolated DNA nucleotide and CNT optimised in vacuum, and (II) using PACs obtained from optimising nucleotide-CNT hybrid in solution. The former approach is what most MD simulations have used in the study of DNA-CNT hybrids, while in the latter approach, a redistribution of the PACs has occurred upon the hybridisation. Our results show that the charge redistribution has a profound effect on the dynamics of binding. In particular, PACs obtained from (II) lead to more stable binding structures in the MD simulations. The findings suggest that care should be taken in simulating DNA-CNT interactions using the classical force field approach.