Irena Peri

Allicin Purified From Fresh Garlic Cloves Induces Apoptosis in Colon Cancer Cells Via Nrf2

Allicin (diallyl thiosulfinate) is the best-known biologically active component in freshly crushed garlic extract. We developed a novel, simple method to isolate active allicin, which yielded a stable compound in aqueous solution amenable for use in in vitro and in vivo studies. We focused on the in vitro effects of allicin on cell proliferation of colon cancer cell lines HCT-116, LS174T, HT-29, and Caco-2 and assessed the underlying mechanisms. This allicin preparation exerted a time- and dose-dependent cytostatic effect on these cells at concentrations ranging from 6.2 to 310 μM. Treatment with allicin resulted in HCT-116 apoptotic cell death as demonstrated by enhanced hypodiploid DNA content, decreased levels of B-cell non-Hodgkin lymphoma-2 (Bcl-2), increased levels of bax and increased capability of releasing cytochrome c from mitochondria to the cytosol. Allicin also induced translocation of NF-E2-related factor-2 (Nrf2) to the nuclei of HCT-116 cells. Luciferase reporter gene assay showed that allicin induces Nrf2-mediated luciferase transactivation activity. SiRNA knock down of Nrf2 significantly affected the capacity of allicin to inhibit HCT-116 proliferation. These results suggest that Nrf2 mediates the allicin-induced apoptotic death of colon cancer cells.

Improving Bioavailability and Stability of Genistein by Complexation with High-Amylose Corn Starch

Genistein, like other phytochemicals, has beneficial health effects, but its bioavailability is limited. This research studied the effect of complexation of genistein with starch on genistein bioavailability. Genistein release from these complexes was tested in vitro under simulated intestinal conditions and in vivo in rats fed high-amylose corn starch (HACS)-genistein complexes (experimental group) as compared to those fed a physical mixture of HACS and genistein (controls). In vitro results showed that genistein release is sustained and fits the normal transit time of food in the intestine. The genistein concentration in the plasma was twice as high in the experimental group versus controls; the genistein concentration in the urine was also higher in the experimental group but lower in the feces. These results indicate that starch-genistein complexes increase genistein bioavailability and suggest that starch can affect the bioavailability of additional food components.

Chemical characterization, antiproliferative and antiadhesive properties of polysaccharides extracted from Pleurotus pulmonarius mycelium and fruiting bodies

Mushroom polysaccharides are potent substances that exhibit antitumor and immunomodulatory properties. Studies comparing the chemical composition and antitumor-related activities of polysaccharides released by fungal strains under different growth conditions are not available. Thus, the present study compared polysaccharides extracts produced by Pleurotus pulmonarius from mycelium grown in liquid culture (ME) or fruiting bodies (FBE). Polysaccharides of both ME and FBE had a relatively high molecular mass. NMR spectroscopy indicated that ME glucan is an α-glucan whereas FBE glucan is a mixture of both α- and β-glucans. Glucose and galactose where the most prominent monosaccharide in both glucans. Treatment of several colon cancer cell lines expressing varying amounts of galectin-3 with the two fungal glucans inhibited their viability and significantly reduced their ability to adhere to the key component of the extracellular matrix, fibronectin, and to a human umbilical vein endothelial cell monolayer, in a time- and dose-dependent manner mainly in those cell lines expressing high amounts of galectin-3. We conclude that ME and FBE glucans may exert a direct antiproliferative effect on cancer cells expressing high galectin-3 concentrations and concomitantly downregulate tumor cell adherence, the latter being directly related to cancer progression and metastasis.

Orally administered glucans from the edible mushroom Pleurotus pulmonarius reduce acute inflammation in dextran sulfate sodium-induced experimental colitis.

Polysaccharides are one of the most potent mushroom-derived substances exhibiting anti-inflammatory and immunomodulatory properties. The aims of the present study were to determine whether orally administered glucans from the edible mushroom Pleurotus pulmonarius could attenuate or prevent the development of experimental colitis in mice. Colonic inflammation was induced in mice by treatment with 3.5 % dextran sulfate sodium (DSS) for 18 d. Before or after DSS administration, mice were given hot water solubles (HWS) or mycelium extract (ME) (2 or 20 mg per mouse) daily in their food. Colonic damage was macroscopically and histologically evaluated. Inflammation was assessed by changes in colon length, TNF-alpha levels released by colonic samples in organ culture and myeloperoxidase (MPO) activity. mRNA levels of pro-inflammatory (IL-1beta) and anti-inflammatory (IL-10) cytokines in colonic samples were determined by quantitative real-time RT-PCR. P. pulmonarius glucans attenuated and prevented the development of symptoms associated with DSS-induced colitis. High doses of HWS and ME blocked colon shortening, suppressed MPO activity and improved macroscopic score in all treatment groups. In addition, histological damage from colitis was reduced by HWS and ME at all doses. The tissue levels of TNF-alpha protein were significantly decreased and correlated with degree of inflammation and macroscopic score. All treatments significantly attenuated the increased DSS-mediated expression levels of IL-1beta. We conclude that the different glucan preparations (HWS or ME) harvested from P. pulmonarius when orally administered to DSS-treated mice attenuate the development of colonic inflammation, suggesting putative clinical utility for these extracts in the treatment of colitis.

PHOSPHOLIPASE D FROM PEANUT SEEDS IV. FINAL PURIFICATION AND SOME PROPERTIES OF THE ENZYME

A procedure for a final purification of phospholipase D from peanut seeds is described. This includes extraction of the seeds, (NH4)2 SO4 fractionation, DEAE-cellulose and Sepharose 6B chromatography followed by preparative disc-gel electrophoresis on polyacrylamide. The purified enzyme yields a single band on analytical disc-gel acrylamide electrophoresis. The following properties of the enzyme were determined: (1) pI value of 4.65; (2) Instability at acidic pH values despite a pH optimum of catalysis of 5.6; (3) Incomplete protection by lecithin from inactivation by acid. Amino acid composition was determined and the N-terminal amino group was found to be glycine. No lipid could be detected in the purified enzyme. Molecular-weight determinations revealed a minimal value of 22 000 ± 3000 using sedimentation equilibrium ultracentrifugation at various pH and temperature values. A molecular weight of 200 000 ± 10 000 was obtained with gel filtration and a value of 48 500 ± 4500 in the presence of denaturing agents such as urea or sodium dodecylsulfate.

Amelioration of diabesity-induced colorectal ontogenesis by omega-3 fatty acids in mice

Postnatal intestinal ontogenesis in an animal model of diabesity may recapitulate morphological and transduction features of diabesity-induced intestinal dysplasia and its amelioration by endogenous (n-3) polyunsaturated fatty acids (PUFA). Proliferation, differentiation, and transduction aspects of intestinal ontogenesis have been studied here in obese, insulin-resistant db/db mice, in fat-1 transgene coding for desaturation of (n-6) PUFA into (n-3) PUFA, in db/db crossed with fat-1 mice, and in control mice. Diabesity resulted in increased colonic proliferation and dedifferentiation of epithelial colonocytes and goblet cells, with increased colonic β-catenin and hepatocyte nuclear factor (HNF)-4α transcriptional activities accompanied by enrichment in HNF-4α–bound (n-6) PUFA. In contrast, in fat-1 mice, colonic proliferation was restrained, accompanied by differentiation of crypt stem cells into epithelial colonocytes and goblet cells and by decrease in colonic β-catenin and HNF-4α transcriptional activities, with concomitant enrichment in HNF-4α-bound (n-3) PUFA at the expense of (n-6) PUFA. Colonic proliferation and differentiation, the profile of β-catenin and HNF-4α-responsive genes, and the composition of HNF-4α-bound PUFA of db/db mice reverted to wild-type by introducing the fat-1 gene into the db/db context. Suppression of intestinal HNF-4α activity by (n-3) PUFA may ameliorate diabesity-induced intestinal ontogenesis and offer an effective preventive modality for colorectal cancer.

Interactions of phospholipase D with 1,2 diacyl-sn-glycerol-3-phosphorylcholine, dodecylsulfate, and Ca2+.

Some properties of the pure, soluble phospholipase D (phosphatidycholine phosphatido hydrolase, EC 3.1.4.4) interactions with phosphatidyl choline (1,2 diacyl-sn-glycerol-3-phosphoryl choline) in a system also containing dodecylsulfate and Ca2+ ions were studied. Concentrations of Ca2+ greater than 50 mM were necessary both for activity and adsorption of the enzyme to the “supersubstrate.” Ethylenediamine tetraacetic acid caused inhibition of activity, greater than one would expect from its chelating capacity. A nonlinear increase in activity with the increase of enzyme protein was observed, suggesting a subunit aggregation into a higher mol wt protein, catalytically more active. Upon centrifugation of the supersubstrate-enzyme complex at 4.5×105g·min at 30 C, most of the substrate molecules sedimented regardless of the pH. The reverse was true when centrifugation was done at 1 C. Phospholipase D hydrolyzed phosphatidylcholine molecules present in the supersubstrate at temperatures around 0 C at a rate 1/5 that of a maximal value measured at 30C. The Arrhenius plot was linear in the range from 0 to 30 C, and at that temperature the curve broke with a smaller slope. Activation energy of 9.1 Kcal/mol, below 30 C, was calculated. Adsorption of the enzyme to the sedimentable supersubstrate occurred at pH 8.0, regardless of temperature. At pH 5.6, a considerable portion of phosphatidylcholine was degraded at 30 C, thus minimizing the capacity of the supersubstrate to adsorb the enzyme. Although Mg2+ could replace Ca2+ in the formation of sedimentable supersubstrate, it neither assists in adsorption of the enzyme nor in activation of the phosphatidylcholine hydrolysis.

Partial purification and characterization of putative paracrine/autocrine bovine mammary epithelium growth factors

Abstract Bovine prepartum milk-like secretion (BPMS) was found to be a rich source of mitogenic activity in an in vitro culture of bovine mammary epithelial cells, grown embedded in collagen and in mouse mammary gland-derived cell lines MME-CI and MME-L7. Partial purification of these mitogenic factors revealed that they are most likely novel heparin-binding growth factors (HBGFs) with molecular weights of 19 and 6 kDa. These factors exhibited strong synergism effect with insulin-like growth factor-I and basic fibroblast growth factor, some additive effect with epidermal growth factor but none with fetal calf serum. Mitogenic factors were also found in a conditioned medium obtained from primary cultures of bovine mammary epithelial cells. However, these factors did not bind to heparinagarose columns. Their partial purification by ion-exchange chromatography and gel-filtration revealed that these factors were small proteins of 6 kDa or less. While the origin of these BPMS-derived growth factors is not certain, the conditioned medium-derived activity is of paracrine or autocrine origin.

Epigenetic control of HNF-4α in colon carcinoma cells affects MUC4 expression and malignancy.

BackgroundWe previously found that enhanced expression of hepatocyte nuclear factor 4α (HNF-4α) is associated with hyper-proliferation of colon carcinoma cells. Here, the effect of histone deacetylase (HDAC) inhibitors on proliferation and the expression of HNF-4α and its downstream target genes were assessed in HM7, LS174T, HT29 and Caco-2 colon carcinoma cell lines.ResultsHNF-4α expression was found to vary in the different colon carcinoma cell lines tested, being highest in HM7. Additionally, a direct correlation with proliferation was observed. In HM7 cells, the weak HDAC inhibitor butyrate significantly inhibited the transcription of HNF-4α, its downstream target gene MUC4, and genes associated with proliferation, including the proliferating cell nuclear antigen gene PCNA. siRNA-mediated silencing of HNF-4α exerted an effect similar to butyrate on HM7 cell proliferation. The stronger HDAC inhibitor trichostatin A (TSA) exerted an effect similar to that of siRNA-mediated HNF-4α silencing and, concomitantly, inhibited the expression of the transcription factor gene SP1. Also, siRNA-mediated silencing of HDAC3 and HDAC4 reduced HNF-4α expression. Chromatin immunoprecipitation (ChIP) assays revealed that TSA induces hyperacetylation of histones H3 and H4 and, concomitantly, inhibits SP1 binding to the HNF-4α promoter. Subsequent electromobility shift assays supported these latter findings.ConclusionsHNF-4α transcriptional expression and activity are tightly controlled by epigenetic mechanisms. HDAC inhibitor targeting of HNF-4α may serve as an effective treatment for advanced colon carcinomas, since downstream cancer-associated target genes such as MUC4 are significantly down-regulated by this treatment.

Biosynthesis of triterpenoid sapogenols in soybean and alfalfa seedlings

Abstract By incubation of germinating soybeans with mevalonate-[2- 14 C] (MVA), radioactivity was incorporated into four sapogenols which were identified by TLC. Unequivocal evidence for the identity of three of the four sapogenols was provided by co-crystallization to constant specific radioactivity. The partition of incorporated radioactivity into lipid- and water-soluble fractions and the pattern of radioactivity of individual sapogenols varied with the mode of administering labeled substrates to soybean seedlings, such as incubation of germinating soybeans with MVA-[2- 14 C], immersion of roots into MVA-[2- 14 C] or foliar application of squalene-[ 14 C]. When alfalfa seedlings were incubated with MVA-[2- 14 C], about two-thirds of the radioactivity incorporated into the sapogenols was associated with medicagenic acid.