Betty Schwartz

Leptin promotes motility and invasiveness in human colon cancer cells by activating multiple signal-transduction pathways

Leptin serum levels are about 5 times higher in obese people than in normal individuals. We aimed at investigating the signaling pathways induced by leptin in the human colonic cell lines LS174T and HM7. Both cells expressed the leptin transmembrane Ob‐receptor. Leptin activated the mitogen‐activated protein kinase pathway, induced invasion of colonic cells and concomitantly increased the formation of lamellipodial structures. A direct and novel dose‐ and time‐dependent activation of RhoA, Cdc42 and Rac1 by leptin is demonstrated in these aggressive colon cancer cells. The activation of the Rho family of GTPases was amenable to specific inhibition: Wortmannin inhibited leptin‐induced Rac1 and Cdc42 activation but did not affect RhoA activation, and inhibited the formation of leptin‐induced lamellipodia and cell invasion. The Rac1 inhibitor NSC23766 inhibited only leptin‐induced Rac1 activation and concomitantly, lamellipodium formation and cell invasion. The Src kinase inhibitor II (SrcKI‐II) exerted a positive effect on RhoA activation, inhibited tyrosine phosphorylation of p190RhoGAP and inhibited leptin‐induced Cdc42 activation and leptin‐induced lamellopodium formation and cell invasion. The specific JAK2 inhibitor AG490 exerted a positive effect on Rac1 and Cdc42 activation by leptin and concomitantly inhibited RhoA activation. AG490 did not inhibit leptin‐induced lamellopodium formation or cell invasion. Our findings clearly indicate that leptin activates PI3K and Src kinase pathways in the metastatic colon cancer cells LS174T and HM7. These signaling pathways induce the activation of Rac1 and Cdc42, lamellopodium formation and concomitantly enhanced cell invasion, but leptin activation of RhoA is not associated with enhanced cell locomotion and invasion. Understanding in‐depth the pathways involved in leptin‐associated enhanced cell locomotion and invasion may contribute with the design of novel therapeutics to treat obesity‐associated advanced colorectal cancer.

Apoptosis cascade proteins are regulated in vivo by high intracolonic butyrate concentration: correlation with colon cancer inhibition.

The present study was aimed at evaluating the effect of high intracolonic butyrate concentrations, either through fermentation of a soluble fiber-enriched diet or via intracolonic butyrate instillation, on colon cancer in a chemically induced (dimethylhydrazine) rat model. The effects were tested in four groups of dimethylhydrazine-treated rats: (i) rats fed a standard diet, (ii) rats fed a diet enriched with 15% citrus pectin, a soluble fiber that ferments and produces a high concentration of intracolonic butyrate, (iii) rats fed a standard diet and intrarectally instilled with a sodium butyrate solution (50 mM), (iv) rats fed a standard diet and intrarectally instilled with sodium butyrate vehicle solution (100 mM NaCl). The apoptotic index in the distal colon of rats fed pectin was higher than in colonic tissue from rats fed a standard diet. The expression of caspase-1, a cysteine protease implicated in the regulation of programmed cell death, as detected by both Northern and Western analysis, showed the highest mRNA and protein levels in colonic tissue from rats intrarectally instilled with butyrate. Immunohistology confirmed the Western blot findings. Expression of the cleaved poly(ADP-ribose) polymerase product, a downstream nuclear substrate for caspase-3 in the apoptotic pathway, was elevated in both the pectin-fed and butyrate-instilled groups. Expression of the antiapoptotic protein Bcl-2 was significantly reduced following pectin feeding as well as butyrate instillation. The highest expression of Bcl-2 was observed in tumor tissue. A marked reduction in aberrant crypt number was observed in colonic tissue obtained from both the pectin-fed and butyrate-instilled groups relative to rats from the standard diet group. The average tumor volume per rat in both the pectin-fed and butyrate-instilled groups was significantly lower than in rats from the standard diet and the sodium butyrate vehicle-instilled groups. We conclude that high butyrate levels, either instilled or obtained following fermentation of soluble dietary fibers, inhibit early and late events in colon tumorigenesis by controlling the transcription expression and activity of key proteins involved in the apoptotic cascade.

Generation of the potent anti-malarial drug artemisinin in tobacco

volume 29 number 12 DeCember 2011 nature biotechnology To the Editor: The emergence of multidrug-resistant strains of Plasmodium spp., the etiological agent of malaria, constitutes a major threat to controlling the disease1,2. Artemisinin, a natural compound from Artemisia annua (sweet wormwood) plants, is highly effective against drug-resistant malaria. Even so, lowcost artemisinin-based drugs are lacking because of the high cost of obtaining natural or chemically synthesized artemisinin1,2. Martin et al.3 were the first to report the generation of an artemisinin precursor in a microbial system. They engineered Escherichia coli with a synthetic mevalonate pathway from Saccharomyces cerevisiae. Expression of amorphadiene synthase (ADS) from A. annua in this strain allowed production of amorpha4,11-diene, the sesquiterpene olefin precursor to artemisinin. However, despite extensive effort invested in the past decade in metabolic engineering of artemisinin and its precursors in both microbial and heterologous plant systems2–6, production of artemisinin itself has never been achieved. Here we report the metabolic engineering of tobacco to produce artemisinin, generating transgenic plants that express five plantand yeast-derived genes involved in the mevalonate and artemisinin pathways, all expressed from a single vector. Our experiments demonstrate that artemisinin can be fully biosynthesized in a heterologous (that is, other than A. annua) plant system, such as tobacco. Although the artemisinin levels we have generated in transgenic tobacco are currently lower than those in A. annua, our experimental platform should lead to the design of new routes for the drug’s commercial production in heterologous plant systems. The World Health Organisation (WHO; Geneva) promotes the use of artemisinin as a first-line treatment for malaria, and it is heavily involved in facilitating the development of artemisinin-based anti-malaria drugs1. Artemisinin is biosynthesized from terpenoid backbones generated by the mevalonate and methyl-erythritol phosphate (MEP) pathways7–9 (Fig. 1a). Although detailed knowledge of the artemisinin-biosynthesis pathway is still lacking, it initiates with the cyclization of farnesyl diphosphate by ADS to form amorpha-4,11-diene, which is then oxidized by the cytochrome P450 CYP71AV1, reduced by artemisinic aldehyde reductase (DBR2) and possibly reoxidized by aldehyde dehydrogenase to yield dihydroartemisinic acid—the presumed precursor of artemisinin in plants6,7,10. Normally, dihydroartemisinic acid accumulates in A. annua and slowly converts to artemisinin, a process that can be stimulated after harvest by drying in the sun. The transformation of dihydroartemisinic acid to artemisinin in A. annua has been proposed to be nonenzymatic, requiring only the presence of light and molecular oxygen6,7; a singlet oxygen formed as a consequence of exposure to UV/visible light may react with dihydroartemisinic acid to form a ketoenol, which can then react with ground-state oxygen to form a second hydroperoxide that spontaneously forms artemisinin. Despite the great interest in enhancing yields of artemisinin in its host A. annua, classic breeding and genetic engineering strategies have met with only limited success6,11. Moreover, recent attempts to produce the artemisinin precursors artemisinic and dihydroartemisinic acids in heterologous plants4,5 did not lead to their accumulation due to internal glycosylation and insufficient oxidation toward the acids. Harnessing E. coli and S. cerevisiae has allowed the production of high titers of amorpha-4,11diene and artemisinic acid2,3, but not of the active artemisinin drug itself. To reconstruct the artemisinin-producing pathway in Nicotiana tabacum, we first generated a mega-vector carrying cytochrome P450 reductase (CPR) from A. annua to prevent the accumulation of inactive oxidized P450, as well as ADS, CYP71AV1 and DBR2 (Fig. 1b and Supplementary Methods). The mega-vector also contained a truncated and deregulated 3-hydroxy-3-methylglutarylcoenzyme A reductase (tHMG) from yeast to increase the supply of precursor from the mevalonate pathway for artemisinin production. To ensure genetic stability and Generation of the potent anti-malarial drug artemisinin in tobacco

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.

A protective role of dietary vitamin D3 in rat colon carcinogenesis.

The aim of the present work was to gain insight into a putative anticancer effect of dietary vitamin D3 (cholecalciferol) in a rat model of colon carcinogenesis. Male rats were assigned to three different dietary groups. The dietary regimens were based on a standard murine-defined diet (AIN-76A) or a stress diet containing 20% fat, reduced Ca2+ concentration, a high phosphorus-to-Ca2+ ratio, and either low or high vitamin D3 content. Colorectal cancer was induced by administration of the procarcinogen 1,2-dimethylhydrazine (DMH). Blood Ca2+, 1,25-dihydroxyvitamin D3 [1,25(OH)2 D3 ], and 25-hydroxyvitamin D3 [25(OH)D3 ] levels were measured in DMH-treated rats and in respective weight- and age-matched dietary control groups. Colonic epithelial proliferation was assessed by determining thymidine kinase (TK) activity, bromodeoxyuridine (BrdUrd) incorporation into crypt cell DNA, and the mean labeling index along the colonic crypt continuum. Maintenance of rats on the stress diet either unmodified or supplemented with vitamin D3 in the absence of carcinogen treatment provoked a time-dependent rise in colonic TK activity and hyperproliferation of colonic epithelium. DMH treatment of rats maintained on the standard diet caused a marked increase in the proliferative indexes of colonic epithelium and in expansion of the crypt proliferative compartment. TK activity and the crypt mitotic zone were significantly augmented in the animal group fed the stress diet. Supplementary vitamin D3 abrogated the stress diet-enhanced colonic responses to the carcinogenic insult. Colon tumor multiplicity was fourfold higher in animals fed the stress diet than in animals maintained on a standard diet. The marked rise in colonic tumor multiplicity and adenocarcinoma incidence in rats fed the stress diet was obliterated by supplemental dietary vitamin D3 Cumulatively, the present results indicate that dietary vitamin D3 impedes the neoplastic process in murine large intestine and strengthen the view that inappropriate changes in dietary components and micronutrients are contributory determinants of colorectal cancer.

Butyrate regulates E-cadherin transcription, isoform expression and intracellular position in colon cancer cells

Cell-to-cell adhesion, an important event in differentiation, is impaired during advanced stages of tumorigenesis. In this study, we examined the possible regulation of cell-adhesion proteins by the differentiation agent butyrate in LS174T and HM7 cells, two types of human colon cancer cells that differ in their ability to produce mucin and colonize the liver of experimental animals. The more aggressive, high-mucin-producing cell line (HM7), a clone selected from LS174T cells, showed a scattered and undifferentiated ultramorphological appearance and low basal alkaline phosphatase activity; the proteins β-catenin and E-cadherin, as detected by immunostaining, were expressed in the cells’ nuclei. All of these properties were significantly less pronounced in the less aggressive, low-mucin-producing LS174T cells. In both cell lines, butyrate treatment enhanced cell-to-cell interaction, alkaline phosphate activity, translocation of β-catenin and E-cadherin from the nuclei to the membrane junctions, and transcription and translation of the 120-kDa E-cadherin isoform, but not of its 100-kDa isoform. Analysis of possible mechanisms of E-cadherin up-regulation revealed that butyrate induces the release of nuclear proteins from the E-cadherin promoter sequence, reducing transcription repression. We suggest that butyrate activates E-cadherin transcription through translocation of nuclear transcription factors bearing specific repressor activity. We surmise that abrogation of nuclear 100-kDa E-cadherin and β-catenin expression following butyrate treatment is related to the control of E-cadherin gene transcription.

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.

A recombinant human RNASET2 glycoprotein with antitumorigenic and antiangiogenic characteristics: expression, purification, and characterization.

Human RNASET2 is a T2‐RNase glycoprotein encoded by the RNASET2 gene, which is located on chromosome 6 (6q27). Deletion in 6q27 is associated with several human malignancies.