Flavonoids, dietary-derived inhibitors of cell proliferation and in vitro angiogenesis

Consumption of a plant-based diet can prevent the development and progression of chronic diseases associated with extensive neovascularization, including solid malignant tumors. In previous studies, we have shown that the plant-derived isoflavonoid genistein is a potent inhibitor of cell proliferation and in vitro angiogenesis. In the present study, we report that certain structurally related flavonoids are more potent inhibitors than genistein. Indeed, 3-hydroxyflavone, 3',4'-dihydroxyflavone, 2',3'-dihydroxyflavone, fisetin, apigenin, and luteolin inhibited the proliferation of normal and tumor cells, as well as in vitro angiogenesis, at half-maximal concentrations in the low micromolar range. We have previously demonstrated that genistein concentrations in the urine of subjects consuming a plant-based diet is 30-fold higher than in subjects consuming a traditional Western diet. The wider distribution and the more abundant presence of flavonoids in the plant kingdom, together with the present results, suggest that flavonoids may contribute to the preventive effect of a plant-based diet on chronic diseases, including solid tumors.     

Measurement of tritiated thymidine labeling index by incubation in vitro of surgically removed cervical cancer

Autoradiographic study was made on 24 cases of uterine cervical tumor, including 22 cases of squamous cell carcinoma, using a technique of in vitro labeling of surgical specimens with tritiated thymidine (3H-TdR) under hyperbaric oxygen. Labeling index of the cervical cancer ranged from 8.7 to 30.4% and was well correlated with the histological subtypes (spinal, transitional, and basal cell types). The upper limit of the growth fraction was also estimated from the general relationship between phase lengths. The necessity of sufficient oxygen tension for the S-phase cells to effect synthesis of DNA was stressed.     

Measurement of DNA adducts in humans after complex mixture exposure

In contrast to acute or chronic dosing experiments with a single chemical in animals, man is exposed to thousands of chemicals during a lifetime. Each of these may act alone, additively, synergistically or antagonistically in terms of biological effects, but most current risk assessment procedures fail to recognize such interactions. In carcinogenesis, a mutational process that is thought to occur through DNA damage by endogenous and/or exogenous agents, a wide variety of host factors is involved in disease outcome. These include absorption of chemicals, their distribution, metabolism and excretion. In addition, once metabolic activation has occurred, there is an array of protective mechanisms that cells have evolved to maintain DNA integrity, such as DNA repair, genetic redundancy and programmed cell death. One approach to risk assessment is to regard all DNA-damaging events as potentially leading to cancer and to measure DNA damage as the biologically relevant endpoint. The main method, if not the only method, presently available to assay a wide range of DNA adducts is 32P-postlabelling. This method has high sensitivity (limit of detection > 1 adduct per 10(10) nucleotides) and is capable of visualizing many different DNA adducts in a single analysis. Postlabelling is best suited for detecting hydrophobic adducts--low molecular weight adducts usually need a preliminary separation procedure prior to being postlabelled. This chromatographic procedure has been used to study DNA samples from human tissues of cigarette smokers, occupationally exposed groups and individuals living in polluted environments. Correlations have been found between the severity of exposure and the level of DNA adducts detected for human samples. However, most studies are single-time point studies, whereas for risk assessment purposes it may be better to use more quantitative and representative measures of long-term exposure, for example the number of adducts formed per annum. This article reviews methods of DNA adduct measurement, with particular reference to the 32P-postlabelling technique, which has been used to determine DNA adduct levels in populations exposed to complex mixtures.     

Detection of Listeria monocytogenes in humans, animals and foods

The Listeria monocytogenes-carrying rates were 100% for listeriosis patients and 1.3% for healthy humans. The L. monocytogenes contamination rates for retail sliced beef (34.2%) and pork (36.4%) were significantly higher (p < 0.05) than those for cattle (2.0%) and pigs (0.8%) and for cattle (4.9%) and swine (7.4%) carcasses. The percentages of serotypes 1/2a, 1/2b and 4b which are most dominant in human patients were high in isolates from fresh (90.0%) and processed (100%) fish and shellfish and imported natural cheese (96.7%).     

Symposium overview: the use of delayed matching-to-sample procedures in studies of short-term memory in animals and humans

The authors examined the hypothesized association between the body burden of polychlorinated biphenyls (PCB) in women and the risk of low birth weight for their infants. In Sweden, a main exposure route for PCBs and other persistent organochlorine compounds is through the consumption of fatty fish from the Baltic Sea (on the Swedish east coast). A previous comparison between a cohort of consumers of large quantities of fish from the Swedish east coast and a reference population, together with a following analysis based on questionnaire data from a case-control study within the east coast cohort, supported the hypothesized association. In 1995, blood samples were collected from the wives and ex-wives of fishermen from the Swedish east coast (n = 192) who had given birth during the period 1973-1991. Cases (n = 57), i.e., infants with low birth weight (1,500-2,750 g), were matched with controls (n = 135; birth weight, 3,250-4,500 g) on gender, parity, and calendar year of birth. The concentration of 2,2',4,4',5,5'-hexachlorobiphenyl (CB-153) in plasma was analyzed; it has been suggested that CB-153 is a relevant biomarker of exposure to PCBs. The concentration of CB-153 in the plasma of mothers during the year of childbirth was "estimated" using some alternative plausible kinetic models. For two alternative estimated exposure datasets, which were focused on separately, an increase in the risk of a low birth weight was observed at a CB-153 concentration of 300 and 400 ng/g lipid weight, respectively (adjusted odds ratios of 2.1 (95% confidence interval (CI) 1.0-4.7) and 2.3 (95% CI 0.9-5.9)). The present results strengthen the findings reported previously for this study population.     

Multiparasite communities in animals and humans: frequency, structure and pathogenic significance

A separate analysis of ulnar and radial finger ridge-counts, obtained from 115 Aymara Indians (55 males and 60 females) of northern Chile, was performed. From these variables, directional asymmetry, fluctuating asymmetry, indices of bilateral asymmetry (square root of A2), and intraindividual diversity (s/square root of 5) were calculated for each sex. The results show that most bimanual differences for the ridge-counts are not statistically significant in the Aymara, except for radial counts in female first and second fingers (right hand means are larger), while most ulnar-radial differences are highly significant in both sexes (radial values exceed ulnar ones). Most sex differences do not reach statistical significance, although males have more ridge-counts, lower directional asymmetry, somewhat lower fluctuating asymmetry, and lower indices of asymmetry and diversity than females. As fluctuating asymmetry is not larger in males, the dermatoglyphic findings do not indicate support for the hypothesis that males are less canalized than females. In accordance with the findings of other authors, interpopulation comparisons in the indices of asymmetry and diversity show ethnic differences. Both indices tend to be low in samples of African ancestry, high in samples of European origin, and intermediate in the Aymara, while Indian groups are characterized by high asymmetry and low diversity values. Moreover, the data reveal a geographical trend in that asymmetry and diversity values tend to decrease from the northern to the southern hemisphere in populations of Europe, the Middle East, and Africa, thus indicating greater ridge-count variability and heterogeneity among fingers in northern populations. It is assumed that this gradient primarily reflects different degrees of miscegenation and heterozygosity.     

An observation of the effect of tanshinone on cancer cell proliferation by Brdu and PCNA labeling

The aim of this study was to find out the anti-cancer activity of Tanshinone and its mechanism of action. Human hepatic carcinoma cell line (SMMC-7721) and leukemia cell line (HL60) were treated with tanshinone the cancer cell proliferation indices were measured by Brdu Labeling and immuno-histochemical stain of PCNA. The Brdu labeling rates of human hepatic carcinoma and leukemia cells treated with tanshinone were 8.95% and 19.01%, which were lower than those of controls (28.0%, 25.57%) respectively (P < 0.01), PCNA positive rates were 57.0% and 30.32%, which were significantly lower than those of controls (74.3%, 47.05%) (P < 0.01). The results indicate that Brdu labeling and PCNA detection may have important utility in the studies of tumor cell proliferation and the relative factors affecting the cell proliferation. The inhibitory effect of tanshinone on cancer cell proliferation might be associated with inhibiting DNA synthesis, PCNA expression and activity of DNA polymerase delta of the tumor cells.     

Suppression of human cancer cell proliferation by lipoxygenase inhibitors and gamma-radiation in vitro

The effects of inhibitors of arachidonic acid oxidative metabolism, gamma-radiation and/or their combinations on proliferation and cell cycle were studied in human breast carcinoma HS578T and monoblastoid U937 cell lines. While piroxicam an inhibitor of cyclooxygenase pathway, had no significant effects on cell proliferation, inhibitors of lipoxygenase pathway, nordihydroguaiaretic acid and esculetin, suppressed [3H]-thymidine incorporation and cell growth. The latter agents also differed in their modulation of cell cycle parameters depending on the cell line and the time of treatment. When the cells were preirradiated with gamma radiation (5 Gy) and treated with the drugs (at concentrations 50 mumol/l and higher) the effects on cell proliferation were mostly additive. On the other hand, the results suggest that antiproliferative effects could be significantly strengthened when lower doses (25 mumol/l) of lipoxygenase inhibitors were combined with a low dose (1 Gy) of gamma-radiation. Experiments monitoring the reversibility of the effects after single or combined treatment with the agents showed that irradiation suppressed the ability of U937 cells to restore cell proliferation, and that these effects may be strenghtened by esculetin. In conclusion, our results (1) suggest that the lipoxygenase pathway plays a significant role in proliferation of cancer HS578T and U937 cells in vitro, and (2) implicate the possibility of more effective antiproliferative effects after combined treatment of cells with gamma-radiation and lipoxygenase inhibitors.     

Methodological findings and considerations in measuring colorectal epithelial cell proliferation in humans

The methodological issues for measuring colorectal epithelial cell proliferation, an intermediate end point for studies of colon neoplasia, in epidemiological studies are deceptively numerous and complex, with few methodological data available. Accordingly, during our experience with measuring colorectal epithelial cell proliferation from nearly 500 participants attending over 1300 study visits over a 6-year period, we recorded data on a variety of measurement variations. Methods investigated included rectal biopsy technique, general histological and labeling procedures [including the tritiated thymidine, 5-bromodeoxyuridine (BrdUrd), and the proliferating cell nuclear antigen (PCNA) immunohistochemical techniques used to label S-phase cells in colonic crypts in rectal biopsy specimens], biopsy scoring procedures, and summary scoring methods. Findings include that the PCNA technique was the simplest, most economical, and least time-consuming. The BrdUrd labeling failure rate was 15% versus < 1% for PCNA. The percentage of labeled cells (labeling index) was highest using PCNA in biopsies processed without prior incubation, intermediate using PCNA in biopsies processed with prior incubation as for BrdUrd, and lowest using BrdUrd. The percentage of labeled cells that were in the upper 40% of the crypt (phi h) was higher using BrdUrd than PCNA; visit-to-visit correlations were higher using PCNA (r = 0.51 versus 0.35), and visit-to-visit variability was lower and between-person variability was higher using PCNA. Intra- and inter-rater reliabilities for the techniques were comparable (PCNA intra-rater r = 0.93, inter-rater r = 0.92). The PCNA technique, compared to the BrdUrd technique, is more feasible and reliable, provides a more accurate estimate of the labeling index, and cell proliferation measures determined with PCNA have statistical properties that are generally more favorable for detecting differences in clinical trials. Thus, the PCNA technique may be preferable to techniques requiring incubation of biopsies. Other methodological findings lead us to recommend that, for larger studies measuring colorectal epithelial cell proliferation on outpatient rectal biopsies, biopsies should be taken 10 cm above the anus using a flexible, preferably jumbo cup, endoscopic forceps through a rigid sigmoidoscope, and histological sections should be 3 microns thick taken 50 microns apart.     

Effects of milk and milk products on rectal mucosal cell proliferation in humans

The interferon (IFN)-induced protein kinase (PKR) functions as a gatekeeper of mRNA translation initiation and is, therefore, a key mediator of the host IFN-induced antiviral defense system. Many viruses have invested countermeasures against PKR. Some apparently use more than one mechanism. The influenza virus can repress PKR activity through the use of at least two factors, the cellular P58IPK protein and the viral NS1 protein. The exact mode of action of the latter has not been established. Here, using a coprecipitation assay, we found that PKR could form a complex with NS1 in crude cell extracts prepared from influenza virus-infected HeLa cells. The NS1-PKR interaction was verified by using the yeast two-hybrid system and an in vitro binding assay. Deletion analysis mapped the NS1 binding site to the N-terminal 98 residues of PKR regulatory region. Furthermore, an NS1 mutant, which lacks PKR inhibitory activity, did not bind PKR. Finally, the functional role of NS1 in PKR inhibition was substantiated using an in vivo assay for PKR activity. These results support the role of NS1 in PKR modulation during viral infection that is mediated through a complex formation between the two proteins.     

Biotransformation of linogliride, a hypoglycemic agent in laboratory animals and humans

Following oral administration of linogliride, a hypoglycemic agent, to rat (50 mg kg-1), dog (30 mg kg-1), and man (100 mg per subject), plasma, urine, and fecal extract sample pools were obtained. Nine metabolites plus unchanged linogliride were isolated and identified. The number of metabolites identified were: rat (5), dog (9), and man (1). In each species, more than 78% of the administered dose was recovered in the urine pools. Identified metabolites were estimated to account for > 82% of the total amounts of drug-related sample in urine pools and > 50% in plasma and fecal extract pools. Formation of linogliride metabolites in the three species can be described by four proposed pathways: pyrrolidine hydroxylation, aromatic hydroxylation, morpholine hydroxylation, and imino-bond cleavage. Comparison of the proposed metabolic pathways among species reveals a similarity between rat and dog. In these two species, pyrrolidine hydroxylation was quantitatively the most important pathway, with 5-hydroxylinogliride and dominant hypoglycemic active metabolite in all sample pools. Further oxidation of 5-hydroxylinogliride resulted in the formation of five minor metabolites. The other three pathways appeared to be quantitatively unimportant. Metabolism of linogliride in man occurred to a very limited extent. More than 90% of the total linogliride-related material in plasma was the unchanged drug. Greater than 76% of the administered dose was excreted unchanged in the urine. Only 5-hydroxylinogliride was identified in minor amounts in human samples.     

Cell density downregulates DNA synthesis and proliferation during osteogenesis in vitro

The classical models of in vitro cell culture comprise fibroblasts and epithelial cells. Osteogenic cells represent another interesting cell model; however, it is not known whether during osteogenesis cell density regulates cell growth as seen in cultures of fibroblasts and epithelial cells. We selected MC3T3-E1 cells for study because they are an osteogenic cell line that, when subcultured, grow to confluence and form multilayers of cells in conventional cultures by continued proliferation, as do fibroblasts. Once maximum cell density is obtained, proliferation is down regulated resulting in a mixed population of quiescent and dividing cells. We used this model to determine whether downregulation of proliferation as expressed by cell number and DNA synthesis is cell density-dependent. MC3T3-E1 cells were cultured over a period of 34 days to determine their kinetics, viability, ability to synthesize DNA, distribution within phases of the cell cycle and cell number-response relationships. Our results show that (1) viability ranged between 92% and 96% and the cell number 2.5 x 10(5) per cm2 once cultures reached steady state, (2) most cells entered the G0/G1 phase of the cell cycle on day 7, (3) there was no correlation between the proportion of cells in S phase and downregulation of DNA synthesis, (4) a direct relationship exists between cell density and downregulation of DNA synthesis on day 8, (5) the minimum time for cells to be cultured before downregulation of DNA synthesis begins is independent of cell number, and (6) downregulation of DNA synthesis is reversible. These results suggest that density-dependent downregulation of DNA synthesis may be a mechanism of growth control for osteogenic cells in vitro that operates more like density-dependent growth control in cultures of fibroblasts rather than epithelial cells.     

Aversively stimulated aggression: Some parallels and differences in research with animals and humans.

On the basis of a review of research with animals and humans, it is argued that a broad range of aversive conditions evoke both flight and fight inclinations. Various factors determine the relative strengths of these dispositions so that the instigation to aggression is not always apparent in overt behavior. It is also maintained that the aversively stimulated aggressive inclination in animals and humans is not only directed toward the diminution of the noxious stimulation (such as pain). Some influences, such as classical conditioning, are common to animals and humans, but thought processes are probably more important in affecting human reactions to aversive events. The role of these thought processes is discussed, and it is suggested that A. Schachter's (1964) cognitive theory of emotions, with its emphasis on the self-labeling of feelings, does not apply to aversively stimulated aggression. A neoassociationistic network conception of emotions is favored as an alternative. It is also suggested that the pain and suffering experienced by depressives contribute to their disposition to aggression. (66 ref) (PsycINFO Database Record (c) 2010 APA, all rights reserved)     

Modulation of glucose production by indomethacin and pentoxifylline in healthy humans

Indomethacin, an inhibitor of prostaglandin synthesis that modulates cytokine production, increases hepatic glucose output (HGO) in humans. However, prostaglandins stimulate glucose production in vitro. To investigate the mechanism of HGO stimulation by indomethacin, we compared the effect of pentoxifylline, an inhibitor of cytokine production, versus saline (study 1, n = 6) and of indomethacin versus the combination of indomethacin and pentoxifylline (study 2, n = 5) on basal HGO. HGO was measured by primed, continuous infusion of 3-3H-glucose. In study 1, pentoxifylline infusion resulted in an immediate, transient decrease of HGO of approximately 50% (from 12.9 +/- 0.4 to 6.0 +/- 1.7 micromol/kg/min after 15 minutes, P < .03 v control). There were no differences in concentrations of glucoregulatory hormones between the two experiments. In study 2, after indomethacin administration, HGO increased transiently by approximately 84% (from 9.7 +/- 0.7 at baseline to 16.7 +/- 2.4 micromol/kg/min after 135 minutes, P < .05). However, pentoxifylline did not affect the increase in HGO induced by indomethacin. There were no differences in concentrations of glucoregulatory hormones between the two experiments. Therefore, indomethacin stimulates HGO by mechanisms unrelated to glucoregulatory hormones, prostaglandins, or cytokines.     

Nicotine stimulates DNA synthesis and proliferation in vascular endothelial cells in vitro

Nicotine is a major component of cigarette smoke and has been postulated to play an important role in atherogenesis and malignancy. Endothelial cell growth may be regulated by nicotine, yet operative mechanisms at the endothelial level are poorly understood. We studied the effects of nicotine (10(-14)-10(-4) M) on endothelial DNA synthesis, DNA repair, proliferation, and cytotoxicity by using cultures of bovine pulmonary artery endothelial cells. Assays were performed on cells incubated with nicotine in the presence and absence of hydroxyurea (an inhibitor of scheduled DNA synthesis), serum, human platelet-poor plasma, and platelet-derived growth factor and endothelial cell growth factor (PDGF and PDECGF, respectively). Nicotine significantly stimulated endothelial cell DNA synthesis and proliferation at concentrations lower than those obtained in blood after smoking (<10(-8) M). The stimulatory effects of nicotine were enhanced by serum (0.5%) and PDECGF and were blocked by the nicotinic-receptor antagonist hexamethonium. The response to nicotine was bimodal because cytotoxicity was observed at higher concentrations (>10(-6) M). This study has implications for understanding cellular mechanisms of nicotine action. The results may be important in tumor angiogenesis, atherogenesis, and vascular dysfunction in smokers.     

Inhibition of human smooth muscle cell proliferation in culture by farnesyl pyrophosphate analogues, inhibitors of in vitro protein: farnesyl transferase

In this study, it was investigated whether and how inhibitors of protein:farnesyl transferase (PFT) can inhibit the proliferation of human smooth muscle cells (HSMC) in culture. Several farnesyl pyrophosphate (FPP) analogues were synthesized and tested in vitro for their specificity in inhibiting squalene synthase (SS), PFT, or protein:geranylgeranyl transferase-1 (PGGT-1) activities (the latter was determined using a newly designed assay). One of these compounds appeared to be a strong PFT inhibitor (IC50 value: 340 nM) and a weak inhibitor in the other two enzyme assays. This compound (designated as TR006) inhibited the farnesylation of Ras in a Ha-ras transfected cell line (Cohen et al., Biochem. Phamacol. 49: 839-845, 1995) and concomitantly slowed down the growth of these cells. Twenty-five microM of TR006 inhibited the proliferation of HSMC isolated from left internal mammary artery, as measured by counting the cells over a period of three cell cycles (10 days). A structurally related compound (TR007), a specific SS inhibitor, did not influence HSMC proliferation under the same conditions. The inhibition by TR006 was concentration-dependent. In HSMC, synchronized by serum depletion, platelet-derived growth factor (PDGF) or basic fibroblast growth factor (bFGF)-induced DNA synthesis was decreased by a 29-hr pretreatment with 100 microM of TR006, indicating that this inhibitor acted in an early phase of the cell cycle, probably by preventing protein isoprenylation. Some other FPP analogues with comparable IC50 values in the in vitro PFT assay were also able to decrease bFGF-induced DNA synthesis without affecting cell viability. A more negatively charged member of this group, TR018, did not influence the growth factor-induced DNA synthesis, probably due to an impaired uptake into the cells. However, the pivaloyloxomethyl derivative of this compound, which is uncharged, and is thought to be converted into TR018 within the cells, showed a strong decrease in bFGF-induced DNA synthesis in HSMC. These data suggest that the compounds investigated may be developed further for treatment of conditions in which undesirable proliferation of smooth muscle cells plays an important role.     

Measurement of DNA strand breakage and DNA repair induced with hydrogen peroxide using single cell gel electrophoresis, alkaline DNA unwinding and alkaline elution of DNA

Three techniques: single cell gel electrophoresis (SCGE), alkaline elution of DNA (AE), and alkaline DNA unwinding (ADU) were chosen to compare the sensitivity among these methods in detection of DNA damage and repair in human diploid VH10 cell line after short-term exposure to hydrogen peroxide. Using SCGE technique a dose-dependent increase in DNA migration was found in cells exposed to hydrogen peroxide in concentration range from 10 micromol/l to 100 micromol/l. Alkaline DNA unwinding method detected increased level of single strand breaks (ssb) in concentration range from 25 micromol/l to 100 micromol/l of H2O2, and alkaline elution of DNA estimated increased DNA elution rate from concentration 50 micromol/l of H2O2. In a time course study to evaluate the kinetics of DNA repair, both SCGE and ADU techniques showed that the repair of DNA strand breaks is very rapid; the level of ssb in treated cells has returned to near the background level within two hours. After this time damage remaining in the DNA was in the form of oxidised bases as revealed the incubation of treated cells with specific DNA repair endonuclease, formamidopyrimidine-DNA glycosylase.     

Overshadowing in landmark learning: Touch-screen studies with pigeons and humans.

Overshadowing in landmark learning was studied in pigeons and undergraduates using a touch-screen spatial search task. Ss searched for an unmarked goal presented in varied locations on a computer screen. Graphic stimuli served as landmarks. The effect of the presence of other landmarks on the control acquired by a given landmark was assessed using a design in which each S was trained with 2 sets of landmarks. Both pigeons (Experiment 1) and humans (Experiments 2–4) showed evidence of learning more about a landmark that was the closest landmark of its set to the goal than about a landmark that was of equal distance to the goal but was not the closest landmark of its set. That is, control by a landmark was overshadowed when it occurred together with a landmark that was closer to the goal. Landmark effectiveness appears to depend not only on the absolute properties of a landmark but on relative factors. The relevance of basic principles of associative learning to spatial landmark learning is discussed. (PsycINFO Database Record (c) 2010 APA, all rights reserved)