Evaluation for safety of antioxidant chemopreventive agents

Antioxidants are considered as the most promising chemopreventive agents against various human cancers. However, some antioxidants play paradoxical roles, acting as "double-edged sword." A primary property of effective and acceptable chemopreventive agents should be freedom from toxic effects in healthy population. Miscarriage of the intervention by beta-carotene made us realize the necessity for evaluation of safety before recommending use of antioxidant supplements for chemoprevention. We have evaluated the safety of antioxidants on the basis of reactivity with DNA. Our results revealed that phytic acid, luteolin, and retinoic acid did not cause DNA damage under the experimental condition. Furthermore, phytic acid inhibited the formation of 8-oxo-7,8-dihydro-2'-deoxyguanosine, an indicator of oxidative DNA damage, in cultured cells treated with a H(2)O(2)-generating system. Thus, it is expected that these chemopreventive agents can safely protect humans against cancer. On the other hand, some chemopreventive agents with prooxidant properties (alpha-tocopherol, quercetin, catechins, isothiocyanates, N-acetylcysteine) caused DNA damage via generation of reactive oxygen species in the presence of metal ions and endogenous reductants under some circumstances. Furthermore, other chemopreventive agents (beta-carotene, genistein, daidzein, propyl gallate, curcumin) exerted prooxidant properties after metabolic activation. Therefore, further studies on safety should be required when antioxidants are used for cancer prevention.     

A method for the prolonged culture of colonic epithelial cells

Summary Many attempts have been made to culture the stem cells that are known to reside at the base of the intestinal crypt. However, no successful method for the long-term culture of these cells has yet been published. We wish to describe a method that maintains human colonic crypt cells in a viable, slowly proliferative state for more than 3 mo. The method involves the use of a viable bovine aortic endothelial cell feeder layer that limits its potential for the study of potential growth factors for the colonic cells.     

A practical prescription for cancer prevention--synergistic use of chemopreventive agents

Enzyme inducers, selenium, and retinoids exert broad-spectrum chemopreventive actions in animal models of carcinogenesis. The mechanisms of action of these three categories of agents are clearly distinct and complementary. BHA, selenium, and beta-carotene are probably safe and non-toxic for humans in doses which, in light of animal studies, can be expected to provide significant cancer protection. The concurrent application of safe, appropriate doses of these three agents could exert a potent synergistic chemopreventive effect, and, if continued throughout life, would in all likelihood substantially reduce cancer risk.     

Gamma interferon-mediated inhibition of Eimeria vermiformis growth in cultured fibroblasts and epithelial cells.

The growth of Eimeria vermiformis within cultured murine fibroblastlike (L-929) or rat epithelial-like (RATEC) cells was inhibited by treatment of the cells with the appropriate recombinant gamma interferon. The effect was apparent as a reduction in both the initial numbers of intracellular sporozoites and, to a much greater extent, the numbers of subsequent developmental stages. Pretreatment of the host cells was more effective than treatment in the early postinvasive period, and recombinant gamma interferon had no effect on the development of the parasite if added 24 h or later after the inoculation of sporozoites. Incubation of sporozoites in medium containing recombinant gamma interferon in no way affected their ability to invade or to grow within host cells. These findings indicate that the inhibitory effects of recombinant gamma interferon on the growth of E. vermiformis are mediated via the host cell and are directed mainly against the transforming sporozoite, although the ability of the sporozoite to invade the host cell was also reduced to some extent. The later developmental stages were refractory to the effects of this lymphokine.     

Assay of growth factors for prostate epithelial cells

Summary The maintenance and proliferation of isolated prostate epithelial cells is androgen independent, but requires multiple other hormones and hormonelike growth factors. Methods are described for isolation and characterization of epithelial cells from normal rat prostate and the androgen-responsive transplantable Dunning R3327 rat tumor. Pure tumor-derived cell lines can be established by serial culture techniques. The normal primary and serially cultured cell lines are then used to assay growth factors. Cell proliferation is quantitated by computerized videometry.     

Isolation and culture of colonic epithelial cells in serum-free medium

Summary Techniques are described for the dissociation, fractionation through Percoll, and in vitro maintenance of mucosal epithelia from the human or guinea pig large bowel. Tissue recovered after surgery is predigested with trypsin-citrate and treated subsequently with a mixture of trypsin, citrate, and collagenase. The resulting suspension of single cells, cell clusters, and partially digested crypts is suspended over a Percoll solution and enriched in multicellular elements by two sequential centrifugations. The recovered multicellular complexes are inoculated to specially treated culture vessels in a serum-free medium supplemented with epidermal growth factor, insulin, transferrin, selenium, and bovine pituitary extract. Epithelia, characterized as such by transmission electron microscopy, adhere to the substrate, form colonies, and can be maintained routinely for study for at least 10 wk.     

Cytotoxicity of Shiga toxin for primary cultures of human colonic and ileal epithelial cells.

Shiga toxin purified from Shigella dysenteriae 1 was cytotoxic to cultured epithelial cells from human colon and ileum. The cytotoxicity, which affected only about 50% of treated cells, was neutralized by rabbit antiserum monospecific for Shiga toxin and mediated by protein synthesis inhibition.     

Induction of NADPH-quinone reductase as a screening assay for potential chemopreventive agents

An in vitro biochemical assay using induction of a phase II enzyme, NADPH-quinone reductase (NADPH-QR) for selection and identification of potential chemopreventive agents is described. A normal human liver cell line (Chang liver cells) or a rodent rat liver cell line (buffalo rat liver cells) was selected as the candidate cell line for induction of QR by a known chemopreventive agent, genistein. The choice of a liver cell line is based on the fact that liver being a major metabolic organ, phase II enzymes are commonly elaborated in liver tissues and is therefore appropriate for enzyme induction studies.     

Development of glutathione induction as a screening assay for potential chemopreventive agents

A simple biochemical assay for screening potential chemopreventive agents using induction of (reduced) glutathione (GSH), in rat liver (BRL-3A) cells is described. BRL-3A cells, cultured to subconfluency, are exposed to potential chemopre-ventive agents for 24 hours and solubilized by sonication. The cytosolic fraction is then assayed for GSH by reacting with O-phthalaldehyde (OPT) to form a fluorescent product that is activated at 350 nm and has an emission peak at 420 nm. The amount of GSH in samples is determined from a regression curve generated from known concentrations of GSH. The percent induction is estimated by comparing the levels in untreated control (basal level) and the agent-treated groups. N-acetyl-l-cysteine at 10 M is routinely included in each assay as a positive inducer of GSH.     

Reversal of inhibition of rat glomerular epithelial cell growth by growth factors.

The ability of several growth factors to reverse heparin-induced inhibition of rat glomerular epithelial cell (GEC) growth and the mechanism of growth inhibition were explored in vitro. Insulin-like growth factor-1, rat multiplication-stimulating activity, and platelet-derived growth factor had no effect on proliferation of cultured GEC exposed to heparin (100 micrograms/ml). Epidermal growth factor (EGF) partially reversed heparin-induced growth inhibition in a dose-dependent fashion with a maximum effect seen at 1 ng/ml. No additive effect was seen with combinations of EGF and the other growth factors assayed. A decrease in EGF-stimulated incorporation of 3H-thymidine by GEC was seen with as little as 2 hours of heparin exposure and persisted for up to 48 hours. Heparin consistently increased binding of 125I-EGF to GEC with a significant increase apparent after 2 hours of exposure and a further increase with a 24-hour exposure. Increased EGF binding to heparin-treated cells was due to a significant increase in the association constant of EGF and its receptor with no effect on receptor number. Interactions between GEC and heparinlike glycosaminoglycans in the glomerular basement membrane may play a role in the regulation of GEC proliferation in normal and diseased states.     

Characterization of acetate uptake by the colonic epithelial cells of the rat

The characteristics of acetate uptake by colonic epithelial cells of the rat were studied. Clear saturation kinetics of acetate uptake were not observed in these experiments at either 0° C or 30° C. A decrease in the pH of the medium markedly increased the acetate uptake. The activation energy for acetete uptake derived from an Arrhenius plot was about 6.1 kcal/mole. Among the inhibitors tested, no effective inhibition of acetate uptake at 0° C was observer. Metabolic inhibitors severely inhibited transport at 30° C. Inhibition of acetate uptake by other short chain fatty acids, which was non-competitive, was observed. The finding that efflux from the cells was stimulated in the presence of compounds such as pyruvate and bicarbonate supported the notion of a close interrelationship between weak electrolyte transports in vivo. Although the H⁺ gradient across the cell membrane is suggested to be one of the factors determining the uptake rate, it seems difficult to explain all the results in this way.     

Synergic production of neutrophil chemotactic activity by colonic epithelial cells and eosinophils

The presence of eosinophils in the lumen and mucosa of the intestine is characteristic of both ulcerative colitis (UC) and Crohn's disease (CD). There is evidence of eosinophil activation in the intestine during acute inflammatory episodes of these diseases; these episodes are also characterized by an influx of neutrophils, which have the potential to cause extensive tissue damage. We undertook a study to determine whether eosinophils in contact with colonic epithelial cells produce factors that may attract neutrophils in response to immunological stimulation. Neutrophil chemotactic activity (NCA) and concentrations of three neutrophil-attracting CXC chemokines – CXCL1 (Groα), CXCL5 (Ena78) and CXCL8 (IL8) – were measured in supernatants of T84 colonic epithelial cells and blood eosinophils or eosinophil-like myeloid leukaemia cells (AML14.3D10), alone or in combination. Cells were stimulated with serum-opsonized zymosan (OZ) particles. NCA (P < 0.005) and CXCL5 levels (P < 0.05) in the supernatants of OZ-stimulated epithelial/eosinophil co-cultures were significantly higher than in the supernatants of either cell type alone. Release of CXCL1 (P < 0.05) and CXCL8 (P < 0.01) from OZ-stimulated co-culture supernatants was significantly higher than from OZ-stimulated eosinophils but not higher than from OZ-stimulated epithelial cells. Eosinophils and colonic epithelial cells exhibit synergy in production of neutrophil chemoattractants in response to immunological stimulation. This may represent a mechanism for exaggerated recruitment of neutrophils to the intestine in response to acute infection in conditions that are characterized by the presence of eosinophils in the bowel.     

Identification of chemopreventive agents by screening for induction of glutathione-S-transferase as a biomarker

For selection and identification of potential chemopreventive agents, a biochemical assay using induction of a phase II enzyme, glutathione-S-transferase (GST) in a liver cell culture is described. A normal human liver cell line (Chang liver cells) was selected as the candidate cell line for induction of GST (liver tissues are abundant in GST) by a known chemopreventive agent, oltipraz. Exponentially growing cells plated for 24 hours are exposed to various doses of a chemopreventive agent for an additional 24 hours, homogenized by sonication, and the homogenate is assayed for GST in a modified microplate enzyme assay using CDNB (1-chloro-2,4-dinitrobenzene) as a substrate. A concurrent protein assay is performed to determine the specific enzyme activity. As the assay is modified from a spectrophotometric assay to a microplate assay, it is reliable, sensitive and fast, a significant number of test agents can be screened in a short time.     

Melanosis coli. A consequence of anthraquinone-induced apoptosis of colonic epithelial cells.

A condition closely resembling human melanosis coli was induced in the guinea pig large intestine by daily oral administration of the anthraquinone danthron. Each treatment caused a transient, dose-related wave of apoptosis of the colonic surface epithelial cells. Most of the resulting apoptotic bodies were phagocytosed by intraepithelial macrophages and carried by them through fenestrae in the epithelial basement membrane to the lamina propria. Here, the apoptotic bodies were transformed into typical lipofuscin pigment in macrophage heterolysosomes. Continued danthron administration caused progressive accumulation of pigmented macrophages in the bowel wall, whereas ongoing migration of pigmented macrophages to regional lymph nodes resulted, after danthron was ceased, in sequential loss of the pigmented cells from the superficial and deep lamina propria. Examination of colonic biopsies from patients with melanosis coli shows increased numbers of apoptotic bodies in the surface epithelium and lamina propria, suggesting implication of the same cellular processes in the formation of the pigment in man.     

VEGF-DsiRNA干扰对人结肠癌细胞增殖及侵袭能力的影响

目的利用人血管内皮生长因子-D(vascular endothelial growth factor-D,VEGF-D)小RNA干扰(siRNA),研究VEGF-D表达下调对人结肠癌细胞增殖和侵袭的影响。方法设立3个细胞组:未处理的人结肠癌SW480细胞作为空白对照组,转染阴性对照siRNA的SW480细胞作为阴性对照组,转染VEGF-D siRNA的SW480细胞作为实验组。RT-PCR和Western-blot检测转染后SW480细胞VEGF-D基因和蛋白的表达,MTT法检测VEGF-D RNA干扰对结肠癌细胞增殖的影响,Transwell小室实验检测VEGF-D RNA干扰对结肠癌细胞侵袭的影响。结果 VEGF-D siRNA转染人结肠癌细胞SW480,VEGF-D基因和蛋白水平表达量明显低于对照组,MTT实验显示体外细胞存活率下降,Transwell小室中穿膜细胞数减少。结论 VEGF-D siRNA显著抑制结肠癌细胞SW480中VEGFD的基因和蛋白表达,明显抑制SW480细胞的体外增殖和侵袭能力。     

Ultrastructural study of Listeria monocytogenes entry into cultured human colonic epithelial cells.

Evidence that Listeria monocytogenes enters Caco-2 cells through the apical surface is presented. Attachment of bacteria to host cells seems to induce modifications of microvilli which are either in direct contact with the bacterial surface or in close vicinity, resulting in the formation of lamellipodia involved in the cellular uptake of the bacteria. Such modifications are not induced by L. monocytogenes SLCC 53, which carries a deletion in the prfA gene, although attachment of this mutant to Caco-2 cells occurs. Listeria innocua does not attach well to Caco-2 cells and also fails to cause structural alterations of the microvilli. Treatment of confluent monolayers of Caco-2 cells with ethylene glycol-bis(beta-aminoethyl ether)- N,N,N1,N1-tetraacetic acid (EGTA), which disrupts intercellular junctions, greatly reduced the uptake of Listeria cells. Attachment and invasion of L. monocytogenes was not accompanied by accumulation of filamentous actin around the entering bacterial cell.