Genetic deletion of mPGES-1 accelerates intestinal tumorigenesis in APC(Min/+) mice

The induced synthesis of bioactive prostanoids downstream of cyclooxygenase-2 (COX-2) and prostaglandin H₂ (PGH₂) exerts a critical event in colorectal carcinogenesis. Here we demonstrate that APC^Min/+ mice with genetic deletion of microsomal prostaglandin E synthase-1 (mPGES-1), which catalyses the terminal conversion of PGH₂ into PGE₂, surprisingly develop more and generally larger intestinal tumors than do mPGES-1 wild type littermates (mean number of tumors/intestine 80 vs. 38, p < 0.0005, mean tumor diameter 1.64 vs. 1.12 mm, p < 0.0005). No deviation regarding the expression of other PGE₂ related enzymes (COX-1, COX-2, mPGES-2, cPGES, and 15-PGDH) or receptors (EP1-4) was obvious among the mPGES-1 deficient mice. PGE₂ levels were suppressed in tumors of mPGES-1 deficient animals, but the concentrations of other PGH₂ derived prostanoids were generally enhanced, being most prominent for TxA₂ and PGD₂. Thus, we hypothesise that a redirected synthesis towards other lipid mediators might (over)compensate for loss of mPGES-1/PGE₂ during intestinal tumorigenesis. Nevertheless, our results question the suitability for mPGES-1 targeting therapy in the treatment or prevention of colorectal cancer.     

Direct single gene mutational events account for radiation-induced intestinal adenoma yields in Apc(Min/+) mice

Data on the induction of small intestinal tumors, predominantly adenomas, by X radiation in Apc(Min/+) mice are reported. Comparison of these incidences with estimates of radiation-induced direct single gene mutation frequencies taken from the literature support the hypothesis that direct mutational loss of Apc+ is the sole requirement for initiation of adenoma. Furthermore, estimates of radiation-induced initiation of adenoma per target stem cell in this animal model are similar to or less than radiation-induced direct somatic gene mutation frequencies. Therefore, while the data reported here do not preclude a role for genomic instability in tumor progression, it is not necessary in this model to postulate the involvement of radiation-induced transmissible genomic instability in initiation of intestinal adenoma.     

Discordant effect of aspirin and indomethacin on intestinal tumor burden in Apc(Min/+)mice

Epidemiologic and animal studies indicate that sustained use of non-steroidal anti-inflammatory drugs (NSAIDs) have a chemopreventive effect against the incidence of colorectal neoplasia and subsequent mortality. We previously demonstrated that sulindac significantly reduces intestinal tumor load in Apc(Min/+)mice and the tumor regression was not necessarily correlated with prostaglandin biosynthesis. In the present study, we further investigate the relationship of NSAID treatment and tumorigenesis in the Apc(Min/+)mouse model. We demonstrate that indomethacin (9 ppm) is a very potent chemopreventive agent, reducing tumor load by 85% and significantly inhibiting basal and ex vivo prostaglandin formation (P< 0.006 and P< 0.0001, respectively). Aspirin (400 ppm) has a similar impact on reducing prostaglandin levels, but in contrast to indomethacin, is uneffective in reducing the tumor load. The data indicate a discordance between the impact of different NSAIDs on tumorigenesis in Apc(Min/+)mice.     

Inhibition of mitosis by avarol, a natural product isolated from the sponge Dysidea avara

Avarol, a sesquiterpenoid hydroquinone, is a cytostatic agent, isolated from the sponge Dysidea avara. Autoradiographic studies show that in vivo (L5178y mouse lymphoma cells) avarol changes the labelling index in favour of the fraction of unlabelled cells (from 1.24 to 1.04). At concentrations below the 50% inhibition dose, the mitotic index increases from 6.5 +/- 0.5 to 10.4 +/- 0.8; at higher concentrations the formation of mitotic figures is almost completely suppressed. In vitro studies applying the methods of viscosimetry and electron microscopy demonstrate that avarol inhibits assembly of brain microtubule protein at an at least stoichiometric concentration ratio. Moreover, evidence is presented that the new antimitotic agent avarol inhibits protofilament elongation rather than lateral association of tubulin during protofilament formation. The results suggest that avarol interferes with polymerization of tubulin both in interphase and during mitosis.     

The natural history of a fugitive prairie plant (Mirabilis hirsuta (Pursh) MacM.)

Summary Some perennial fugitive plants that colonize badger disturbances in xeric prairies have a limited dispersal capacity, and consequently propagules are dispersed over a small area. I hypothesized that high density-dependent mortality might occur early in the life history of such species, and thus increased survival might occur in subsequent age classes because intraspecific competition would be reduced. These hypotheses were tested using natural and experimental cohorts of Mirabilis hirsuta (Pursh) MacM. From these data and field observations, inferences were obtained concerning selective forces operating upon life history characteristics of this species.The distance between individuals of M. hirsuta increases in successive age classes; the greatest decrease in density occurs between the propagule and seedling age classes. Mortality of propagules due to predation by ants and mice was density-dependent. Predation rates were highest at high propagule densities and predation upon propagules located on badger disturbances was higher than the mortality of propagules at similar densities in undisturbed prairie. The results of mortality in the propagule age class are seedlings present only at low densities and located away from parent plants. Seedlings survive to maturity only if they are located on badger disturbances; this species apparently can not successfully compete with plants present in undistrubed prairie. On badger disturbances seedlings present at low densities have much higher survival (roughly 50%) to maturity than do seedlings present at high densities (essentially zero). Thus, if high densities of propagules occur on a disturbance, predation upon propagules results, indirectly, in increased survival of seedlings to maturity. Such predation potentially could have important effects upon interspecific competition of M. hirsuta with other fugitives also colonizing badger disturbances.Reproductive success of M. hirsuta on the Cayler Prairie Preserve is contingent upon successful colonization of disturbance sites. It would appear that selection has operated upon the life history characteristics to favor both successful immigration onto new sites and establishment of seedlings on those sites. Relatively few, but large propagules are produced annually over a long adult life span. While large propagules enhance seedling establishment on xeric sites, production of few propagules annually for a number of years increases the likelihood of immigration onto sites that are variable in the time of appearance within the dispersal range of the plant.     

Inhibition of cyclin-dependent kinase 4 (Cdk4) by fascaplysin, a marine natural product

Small chemical molecules that interfere with biological proteins could be useful for gaining insight into the complex biochemical processes in mammalian cells. Cdk4 is a key protein whose activity is required not only for emergence of cells from quiescence but also at the G1/S transition in the cell cycle and which is misregulated in 60-70% of human cancers. We set out to identify chemical inhibitors of Cdk4 and discovered that, in vitro, fascaplysin specifically inhibited Cdk4. Molecular modelling based on the crystal structure of Cdk2 suggests that fascaplysin inhibits Cdk4 by binding to the ATP pocket of the kinase. Treatment of tumour (p16(-), pRb(+)) and normal (p16(+), pRb(+)) cell lines with fascaplysin caused G1 arrest and prevented pRb phosphorylation at sites implicated as being specific for Cdk4 kinase. Fascaplysin will therefore prove to be a useful tool in studying the consequence of Cdk4 inhibition, especially in cells containing inactivated p16.     

Cyanidin-3-glucoside, a natural product derived from blackberry, exhibits chemopreventive and chemotherapeutic activity

Epidemiological data suggest that consumption of fruits and vegetables has been associated with a lower incidence of cancer. Cyanidin-3-glucoside (C3G), a compound found in blackberry and other food products, was shown to possess chemopreventive and chemotherapeutic activity in the present study. In cultured JB6 cells, C3G was able to scavenge ultraviolet B-induced *OH and O2-* radicals. In vivo studies indicated that C3G treatment decreased the number of non-malignant and malignant skin tumors per mouse induced by 12-O-tetradecanolyphorbol-13-acetate (TPA) in 7,12-dimethylbenz[a]anthracene-initiated mouse skin. Pretreatment of JB6 cells with C3G inhibited UVB- and TPA-induced transactivation of NF-kappaB and AP-1 and expression of cyclooxygenase-2 and tumor necrosis factor-alpha. These inhibitory effects appear to be mediated through the inhibition of MAPK activity. C3G also blocked TPA-induced neoplastic transformation in JB6 cells. In addition, C3G inhibited proliferation of a human lung carcinoma cell line, A549. Animal studies showed that C3G reduced the size of A549 tumor xenograft growth and significantly inhibited metastasis in nude mice. Mechanistic studies indicated that C3G inhibited migration and invasion of A549 tumor cells. These finding demonstrate for the first time that a purified compound of anthocyanin inhibits tumor promoter-induced carcinogenesis and tumor metastasis in vivo.     

Inhibition of tumor growth in mice by endostatin derived from abdominal transplanted encapsulated cells

Endostatin,a C-terminal fragment of collagen 18a,inhibits the growth of established tumorsand metastases in vivo by inhibiting angiogenesis.However,the purification procedures required for large-scale production and the attendant cost of these processes,together with the low effectiveness in clinicaltests,suggest that alternative delivery methods might be required for efficient therapeutic use of endostatin.In the present study,we transfected Chinese hamster ovary(CHO)cells with a human endostatin geneexpression vector and encapsulated the CHO cells in alginate-poly-L-lysine microcapsules.The release ofbiologically active endostatin was confirmed using the chicken chorioallantoic membrane assay.The encap-sulated endostatin-expressing CHO cells can inhibit the growth of primary tumors in a subcutaneous B 16tumor model when injected into the abdominal cavity of mouse.These results widen the clinical applicationof the microencapsulated cell endostatin delivery system in cancer treatment.     

Inhibition of mouse bone marrow granulocyte-macrophage colony formation by factors derived from natural killer cells: an in vitro model for hybrid resistance

Investigations were performed to study whether soluble factors produced by NK-cells could mediate "hybrid resistance" in vitro. NK-cells enriched from spleens of B6D2F1 hybrid mice were incubated with parental B6 bone marrow, and the effect of the derived supernatants on the development of granulocyte-macrophage colony forming cells (GM-CFC) was assessed. Cell free supernatants obtained from low density cells (LDC) of B6D2F1 hybrids stimulated with bone marrow cells (BMC) from B6 mice inhibited GM-CFC formation. The inhibition was similar using B6, D2 or B6D2F1 bone marrow cells as the targets for GM-CFC growth. Our findings suggest that NK cells from F1 hybrid mice when stimulated with BMC from B6 mice release inhibitory factors, different from IFN-gamma and that this production may represent a mechanism of natural resistance to parental H-2b bone marrow grafts.     

Elimination of multidrug-resistant plasmid in bacteria by plumbagin,a compound derived from a plant

Plumbagin (5-hydroxy-2-methyl-1,4-naphthaquinone), a compound derived from the root of thePlumbago zeylanica plant, was effective in selectively eliminating stringent, conjugative, multidrug-resistant plasmids fromEscherichia coli strains. Simultaneous loss of resistance to antibiotics in plumbagin-treated cells indicated loss of plasmid. However, such R plasmids are refractory to treatment with acridine orange and sodium dodecyl sulfate, which are widely used in curing techniques.     

Promotion of adenoma growth by dietary inulin is associated with increase in cyclin D1 and decrease in adhesion proteins in Min/+ mice mucosa

We have earlier shown that dietary fructo-oligosaccharide inulin enhances adenoma growth in multiple intestinal neoplasia (Min/+) mice. To further explore inulin-induced early biochemical changes in the normal-appearing mucosa, Min/+ mice were fed from the age of 5 weeks to the ages of 8 and 15 weeks a control diet or an inulin-enriched diet (10% w/w). In addition, the wild-type littermates were fed with the same diets until the age of 8 weeks, in order to determine whether similar changes happen both in the wild-type and Min/+ mice. The mucosa without adenomas was collected and fractionated to nuclear, cytosolic and membrane pools. The protein levels of beta-catenin, cyclin D1 and E-cadherin were determined by Western blotting at both time points, and immunohistochemical stainings were done for 8-week-old mice. The promotion of adenoma growth by inulin (week 15, 1.3-fold increase, P=.0004) was associated with accumulation of cytosolic and nuclear beta-catenin, and increased amount of cytosolic cyclin D1 (1.5-fold increase, P=.003) in the normal-appearing mucosa of the Min/+ mice. Furthermore, inulin feeding reduced the membranous pools of beta-catenin and E-cadherin. Also in the wild-type mice the drop in membranous beta-catenin was clear (P=.015), and, moreover, a subset of crypts had enhanced nuclear beta-catenin staining. These data indicate that dietary inulin can already activate in the normal-appearing mucosa beta-catenin signaling, which in the presence of Apc mutation induces adenoma growth and even in the wild-type mice direction of the changes is similar.     

Inhibition of interdomain motion in g-actin by the natural product latrunculin: a molecular dynamics study

As part of the cytoskeleton, actin is essential for the morphology, motility, and division of eukaryotic cells. Recent X-ray fiber diffraction studies have shown that the conformation of monomeric actin is flattened upon incorporation into the filament by a relative rotation of its two major domains. The antiproliferative activity of latrunculin, a macrolide toxin produced by sponges, seems to be related to its binding to monomeric actin and inhibition of polymerization. Yet, the mechanism of inhibition is not known in detail. Here, multiple explicit water molecular dynamics simulations show that latrunculin binding hinders the conformational transition related to actin polymerization. In particular, the presence of latrunculin at the interface of the two major domains of monomeric actin reduces the correlated displacement of Domain 2 with respect to Domain 1. Moreover, higher rotational flexibility between the two major domains is observed in the absence of ATP as compared to ATP-bound actin, offering a possible explanation as to why actin polymerizes more favorably in the absence of nucleotides.     

Inhibition of aflatoxin B production of Aspergillus flavus, isolated from soybean seeds by certain natural plant products

AIMS: The inhibitory effect of cowdung fumes, Captan, leaf powder of Withania somnifera, Hyptis suaveolens, Eucalyptus citriodora, peel powder of Citrus sinensis, Citrus medica and Punica granatum, neem cake and pongamia cake and spore suspension of Trichoderma harzianum and Aspergillus niger on aflatoxin B(1) production by toxigenic strain of Aspergillus flavus isolated from soybean seeds was investigated. METHODS AND RESULTS: Soybean seed was treated with different natural products and fungicide captan and was inoculated with toxigenic strain of A. flavus and incubated for different periods. The results showed that all the treatments were effective in controlling aflatoxin B(1) production. Captan, neem cake, spore suspension of T. harzianum, A. niger and combination of both reduced the level of aflatoxin B(1) to a great extent. Leaf powder of W. somnifera, H. suaveolens, peel powder of C. sinensis, C. medica and pongamia cake also controlled the aflatoxin B(1) production. CONCLUSIONS: All the natural product treatments applied were significantly effective in inhibiting aflatoxin B(1) production on soybean seeds by A. flavus. SIGNIFICANCE AND IMPACT OF THE STUDY: These natural plant products may successfully replace chemical fungicides and provide an alternative method to protect soybean and other agricultural commodities from aflatoxin B(1) production by A. flavus.     

Inhibition of D1, D2, and a cyclin expression in HL-60 cells by the lipid peroxydation product 4-hydroxynonenal

4-Hydroxynonenal (HNE), a product of lipid peroxidation, is an highly reactive aldehyde that, at concentration similar to those found in normal cells, blocks proliferation and induces a granulocytic-like differentiation in HL-60 cells. These effects are accompained by a marked increase in the proportion G0/G1 cells. The mechanisms of HNE action were investigated by analyzing the expression of the cyclins and cyclin-dependent protein kinases (CDKs), controlling the cell cycle progression. Data obtained by exposing cells to dimethyl sulfoxide (DMSO) were used for comparison. 4-Hydroxynonenal downregulated both mRNA and protein contents of cyclins D1, D2, and A until 24 h from the treatments, whereas DMSO inhibited cyclin D1 and D2 expression until the end of experiment (2 days) and induces an increase of cyclin A until 1 day. Cyclins B and E, and protein kinase CDK2 and CDK4 expressions were not affected by HNE, whereas DMSO induced an increase of cyclin E, B, and CDK2 from 8 h to 1 day. These data are in agreement with previous results indicating a different time-course of accumulation in G0/G1 phases of cells treated with HNE and DMSO and suggest that the HNE inhibitory effect on proliferation and cell cycle progression may depend by the downregulation of D1, D2, and A cyclin expression.     

Inhibition of microbial growth by ajoene, a sulfur-containing compound derived from garlic.

Ajoene, a garlic-derived sulfur-containing compound that prevents platelet aggregation, exhibited broad-spectrum antimicrobial activity. Growth of gram-positive bacteria, such as Bacillus cereus, Bacillus subtilis, Mycobacterium smegmatis, and Streptomyces griseus, was inhibited at 5 micrograms of ajoene per ml. Staphylococcus aureus and Lactobacillus plantarum also were inhibited below 20 micrograms of ajoene per ml. For gram-negative bacteria, such as Escherichia coli, Klebsiella pneumoniae, and Xanthomonas maltophilia, MICs were between 100 and 160 micrograms/ml. Ajoene also inhibited yeast growth at concentrations below 20 micrograms/ml. The microbicidal effect of ajoene on growing cells was observed at slightly higher concentrations than the corresponding MICs. B. cereus and Saccharomyces cerevisiae were killed at 30 micrograms of ajoene per ml after 24 h of cultivation when cultivation was started at 10(5) cells per ml. However, the minimal microbicidal concentrations for resting cells were at 10 to 100 times higher concentrations than the corresponding MICs. The disulfide bond in ajoene appears to be necessary for the antimicrobial activity of ajoene, since reduction by cysteine, which reacts with disulfide bonds, abolished its antimicrobial activity.     

Resveratrol, a natural product derived from grape, exhibits antiestrogenic activity and inhibits the growth of human breast cancer cells

Resveratrol is a natural phytoalexin compound found in grapes and other food products. In this study, the effect of resveratrol on the growth of human breast cancer cells was examined. Results show that resveratrol inhibits the growth of estrogen receptor(ER)-positive MCF-7 cells in a dose-dependent fashion. Detailed studies with MCF-7 cells demonstrate that resveratrol antagonized the growth-promoting effect of 17-beta-estradiol (E2) in a dose-dependent fashion at both the cellular (cell growth) and the molecular (gene activation) levels. At 5 x 10(-6) M, resveratrol abolished the growth-stimulatory effect mediated by concentrations of E2 up to 10(-9) M. The antiestrogenic effect of resveratrol could be observed at a concentration of 10(-6) M and above. The antiestrogenic effect of resveratrol was also demonstrated at the molecular level. Resveratrol in a dose-dependent fashion antagonized the stimulation by E2 of progesterone receptor gene expression in MCF-7 cells. Moreover, expression of transforming growth factor-alpha and insulin-like growth factor I receptor mRNA was inhibited while the expression of transforming growth factor beta2 mRNA was significantly elevated in MCF-7 cells cultivated in the presence of resveratrol (10(-5) M). In summary, our results show that resveratrol, a partial ER agonist itself, acts as an ER antagonist in the presence of estrogen leading to inhibition of human breast cancer cells.     

X radiation up-regulates the occurrence and the multiplicity of invasive carcinomas in the intestinal tract of Apc(min/+) mice

X rays are well known to cause genetic damage and to induce many types of carcinomas in humans. The Apc(min/+) mouse, an animal model for human familial adenomatous polyposis (FAP), contains a truncating mutation in the APC gene and spontaneously develops intestinal adenomas. To elucidate the role of X rays in the development of intestinal tumors, we examined the promotion of carcinogenesis in X-irradiated Apc(min/+) mice. Forty out of 77 (52%) X-irradiated Apc(min/+) mice developed adenocarcinomas that invaded the proprial muscle layer of the small intestine; 24 of 44 (55%) were in males, and 16 of 33 (49%) were in females. In contrast, invasive carcinomas were detected in the small intestines of only 13 of 64 (20%) nonirradiated Apc(min/+) mice; nine of 32 (28%) were in males and four of 32 (13%) were in females. These differences between X-irradiated and nonirradiated Apc(min/+) mice in the occurrence of invasive intestinal carcinomas were statistically significant (P < 0.05 for males, P < 0.005 for females). In wild-type mice, invasive carcinomas were not detected in either X-irradiated or nonirradiated mice. Apc(min/+) mice had many polyps in the large intestine with or without X irradiation; there was no difference in the number of polyps between the two groups. Also, invasive carcinomas were not detected in the large intestine with or without irradiation. The occurrence of mammary tumors, which was observed in Apc(min/+) mice, was found to be increased in irradiated Apc(min/+) mice (P < 0.01). Apc(min/+) mice had many polyps in the small and large intestines with or without X irradiation. X-irradiated Apc(min/+) mice had highly invasive carcinomas in the small intestine with multiplicities associated with invasiveness. Our results suggest that X radiation may promote the invasive activity of intestinal tumors in Apc(min/+) mice.     

Inhibition of alkaline phosphatase by surfactin, a natural chelating lipopeptide from Bacillus subtilis

Surfactin, an acidic lipopeptide of Bacillus subtilis, at 70 μM non-competitively inhibited alkaline phosphatase without its glycosyl-phosphatidylinositol anchor by 50%. Enzyme activity was restored by adding 50 μM ZnCl ₂ and 1 mM MgCl₂ confirming the chelating action of the free carboxyl groups of the Asp and Glu residues in the peptide moiety of surfactin. This revised version was published online in November 2006 with corrections to the Cover Date.