Ernice Blann

Chemopreventive Effects of Tea Extracts and Various Components on Human Pancreatic and Prostate Tumor Cells In Vitro

Pancreatic and prostate cancers pose serious problems to human health. To determine the potential for chemopreventive intervention against pancreatic and prostate cancers, black and green tea extracts and components of these extracts were examined in vitro for their effect on tumor cell growth. Components included a mixture of polyphenols from green tea (GTP), mixtures of polyphenols (BTP) and of theaflavins (MF) from black tea, and the purified components epicatechin-3-gallate (ECG) and epigallocatechin-3-gallate (EGCG). Two human cell lines, pancreatic adenocarcinoma (HPAC) and prostate tumor (LNCaP), were exposed to these agents for 24 hours. Results showed inhibition (approx 90%) of cell growth in pancreatic tumor cells by black and green tea extracts (0.02%). GTP (10 micrograms/ml) and MF (100 micrograms/ml) significantly inhibited growth (approx 90%); ECG and EGCG inhibited growth as well (approx 95%). Black and green tea extracts, GTP, and EGCG decreased the expression of the K-ras gene, as determined by reverse transcription-polymerase chain reaction. Green and black tea extracts decreased the multidrug-resistant gene (mdr-1), although GTP and EGCG increased expression. Similar data were obtained in the prostate cell line LNCaP. All agents significantly inhibited growth. These agents increased expression of the mdr-1 gene. This study suggests that components from black and green tea extracts can modulate the expression of genes known to play a role in the carcinogenesis process and, therefore, may be potential agents for chemoprevention against pancreatic cancer.

Human Placental Transport of Oxytocin

Oxytocin (OX) has been suggested as a signal for parturition. Although OX is produced by both mother and fetus, concentrations are higher in umbilical than maternal blood. In addition, umbilical artery OX concentrations (15-40 pg/ml) are higher than umbilical vein (4-12 pg/ml) and maternal (1-10 pg/ml) concentrations. The umbilical A-V difference suggests that placental uptake and transport may be one path from fetal (F) to maternal (M) circulation. However, this difference may also reflect placental oxytocinase activity, which is known to metabolize biologically active peptides such as OX. We have investigated placental transport of OX from F to M and M to F circulation using in vitro dually perfused isolated cotyledons from term human placenta. Term human placentae from uncomplicated pregnancies were obtained immediately after delivery. A single peripheral cotyledon and corresponding lobule was cannulated and perfused. After stabilization and demonstration of adequate M to F perfusion-perfusion overlap, we studied the transport of OX (3H) with 14C-inulin (14C-IN) as permeability reference in both M to F (n = 8) and F to M (n = 6) directions during 2 h of perfusion. In addition to the higher tissue uptake observed in M to F than F to M transport direction as measured by the drop in the concentration of both 3H-OX and 14C-IN in the circuits in which both compounds were added, the same trend was found for the transfer rates of both compounds. These transfer rates which reflect the permeability of placental tissue to OX and IN were 15.17 +/- 2.79 (mean +/- SD) and 6.28 +/- 0.93 microliters/min/g (M to F) and 11.79 +/- 1.77 and 4.91 +/- 0.81 microliters/min/g (F to M). Although the permeability of both compounds is higher in the M to F than in the F to M transport direction, comparing these permeability values with respect to their molecular weight (MW) showed a significant correlation when known permeability values of polar compounds between MW 60 and 68,000 daltons were included. This correlation indicates that OX crosses the placenta in both directions by simple diffusion. High-performance liquid chromatography analysis showed that there is little evidence of placental metabolism and degradation of OX over the period of these experiments. Oxytocin is the main therapeutic drug that is frequently used in obstetrics for the induction of labor and parturition. Under such circumstances and with respect to the placental permeability results, oxytocin could reach the fetal circulation.

Differences in hepatotoxicity and gene expression profiles by anti-diabetic PPAR γ agonists on rat primary hepatocytes and human HepG2 cells

SummaryAgonists of peroxisome proliferator-activated receptor γ (PPARγ) are a new class of oral drugs designed to treat insulin-resistant diabetes (i.e., type 2 diabetes). However, troglitazone, the first compound in the class approved by the US Food and Drug Administration (FDA) in 1997 was found to be hepatotoxic and was withdrawn from the market after reports of severe liver failure. The mechanism of PPAR γ agonist-induced hepatotoxicity remains unknown. In this study, we examined the hepatotoxic effects of five PPAR γ agonists (ciglitazone, pioglitazone, rosiglitazone, troglitazone, and JTT-501) on rat primary hepatocytes and human HepG2 cells. We also compared the gene expression profiles of rat primary hepatocytes after exposure to PPAR γ agonists by using the Rat Genome Survey Microarray system from Applied Biosystems in order to understand the mechanisms of hepatotoxicities induced by PPARγ agonists. Consistent with the hepatotoxicity data, our results demonstrate that the gene expression profiles affected by troglitazone and ciglitazone can be clearly distinguished from those by pioglitazone and rosiglitazone. Genes that are differentially expressed between the more toxic troglitazone/ciglitazone group and the less toxic rosiglitazone/pioglitazone group are involved in necrotic, apoptotic, and cell proliferative pathways. The five compounds were also clustered based on a set of molecular descriptors. The clustering based on chemical structural information is in good agreement with the clustering of compounds based on cytotoxicity or gene expression data, indicating a strong relationship between chemical structure and biological endpoints. Our work suggests that microarray analysis together with toxicological observations can be used to rank drugs for hepatotoxicity and to evaluate the safety of new compounds.

Differential gene expression in mouse primary hepatocytes exposed to the peroxisome proliferator-activated receptor α agonists

BackgroundFibrates are a unique hypolipidemic drugs that lower plasma triglyceride and cholesterol levels through their action as peroxisome proliferator-activated receptor alpha (PPARα) agonists. The activation of PPARα leads to a cascade of events that result in the pharmacological (hypolipidemic) and adverse (carcinogenic) effects in rodent liver.ResultsTo understand the molecular mechanisms responsible for the pleiotropic effects of PPARα agonists, we treated mouse primary hepatocytes with three PPARα agonists (bezafibrate, fenofibrate, and WY-14,643) at multiple concentrations (0, 10, 30, and 100 μM) for 24 hours. When primary hepatocytes were exposed to these agents, transactivation of PPARα was elevated as measured by luciferase assay. Global gene expression profiles in response to PPARα agonists were obtained by microarray analysis. Among differentially expressed genes (DEGs), there were 4, 8, and 21 genes commonly regulated by bezafibrate, fenofibrate, and WY-14,643 treatments across 3 doses, respectively, in a dose-dependent manner. Treatments with 100 μM of bezafibrate, fenofibrate, and WY-14,643 resulted in 151, 149, and 145 genes altered, respectively. Among them, 121 genes were commonly regulated by at least two drugs. Many genes are involved in fatty acid metabolism including oxidative reaction. Some of the gene changes were associated with production of reactive oxygen species, cell proliferation of peroxisomes, and hepatic disorders. In addition, 11 genes related to the development of liver cancer were observed.ConclusionOur results suggest that treatment of PPARα agonists results in the production of oxidative stress and increased peroxisome proliferation, thus providing a better understanding of mechanisms underlying PPARα agonist-induced hepatic disorders and hepatocarcinomas.

Increased expression of hepatic DNA methyltransferase in smokers

The DNA methyltransferase enzyme (DNA MTase) catalyzes DNA methylation at cytosines in CpG dinucleotides. 5-Methylcytosine modification of DNA is important in gene regulation, DNA replication, chromatin organization and disease. Increased levels of DNA MTase have been associated with the initiation and promotion of cancer. This study was conducted to assess whether cigarette smoking and other factors, such as age and gender, influence DNA MTase expression in nontumorous tissue. DNA MTase was significantly (p<0.05) higher in samples from cigarette smokers; the mean level of DNA MTase mRNA was almost 2-fold higher in these samples than in those from nonsmokers. Levels of DNA MTase mRNA were higher in samples from females than in those from males, but the difference was not statistically significant. Age was not associated with DNA MTase levels. Increased levels of DNA MTase in individuals who smoke may indicate a greater susceptibility to the risk of cancer since increased levels of this enzyme are found in cancer cell lines and human tumors. The results of this study suggest that further investigations of increased expression of this enzyme as a predisposing factor for cancer susceptibility are needed.

The effects of phytoestrogens on human pancreatic tumor cells in vitro

Diet has been implicated as a possible link to the etiology, promotion and/or progression of many diseases, including cancer. Recently, interest has been focused on the cancer-protective role of several of the hormone-like diphenolic phytoestrogens, lignans, and isoflavonoids. This study examined the chemoprotective effects of genistein, biochanin A, equol, and coumestrol on human pancreatic adenocarcinoma cells in vitro. Two human adenocarcinoma cell lines, HPAF-11 from a male and Su 86.86 from a female, were used. HPAF-11 cells were exposed for 24 h to these agents at concentrations of 1 and 10 microM. Su 86.86 cells were exposed for 24 h at a concentration of 1 microM. Coumestrol and equol at higher concentrations were toxic to the Su 86.86 cells. These agents displayed marked differences between cell lines in inhibition of growth. Equol and coumestrol inhibited the growth of the female pancreatic tumor cells by 95%; however, these agents stimulated the growth of pancreatic tumor cells from the male. Genistein also stimulated growth in the male pancreatic tumor cells, but had little effect on pancreatic tumor cells from the female. Biochanin A inhibited growth of both male and female tumor cells, but to a lesser extent than other agents. This study also indicated a difference in K-ras expression in pancreatic tumors cells treated with these agents. Equol and coumestrol decreased K-ras expression in the female tumor cell line. Genistein increased expression of K-ras in both male and female pancreatic tumor cells. Genistein also increased expressions of the multidrug resistant (mdr-1) gene in the male tumor-cell line, while coumestrol and biochanin A decreased expression. Equol had no effect on mdr-1 expression. Whether the chemoprotective potential of equol and coumestrol against pancreatic cancer is greater in females than males is being further studied.

Impact of cocaine on human placental function using an in vitro perfusion system

The transport of cocaine from the maternal to fetal circulation and the effect of cocaine on placental function was investigated in vitro using dually perfused term human placentae with recirculation of both maternal and fetal perfusates. In the first experimental group (n = 5, 2 hr), after addition of 3H-cocaine and 14C-inulin to the maternal circulation, steady state concentrations were achieved within 20 min on the maternal side. However, in the following 100 min, uptake of 3H-cocaine remained higher than of the 14C-inulin on the maternal side. 3H-Cocaine was transported more rapidly than 14C-inulin into the fetal circulation and was detected within 10-15 min of initiation of perfusion. In the second experimental group (n = 6), the maternofetal permeability of 14C-insulin was determined in the same placental perfusion in both the absence (control period, 2 hr) and presence of cocaine (test phase, 2 hr) with its 3H-tracer. After the addition of cocaine (2-3 mg/L), the transfer of 14C-inulin was reduced from 6.59 to 3.64 mL/gm per min (p < .001), indicating that cocaine alters placental permeability. In addition to its effect on placental permeability, cocaine decreases the rate of release of hCG into the maternal circulation--reduced from 3.11 (control period) to 1.62 IU/min (test phase, p < .01).

Continuous pO2 Monitoring during Dual Perfusion of the Term Human Placenta in vitro

A computerized system has been developed to continuously monitor, collect and display pO2 in real time from the maternal and fetal arteries and veins in the dually perfused term placenta. Oxygen electrodes were installed in flow-through chambers in the tubing of the perfusion system. The signal from the O2 electrodes was digitized, acquired, analyzed and displayed in real time during the perfusions using a personal computer. Output from the O2 electrodes was linearly proportional to pO2 (33-502 mm Hg; r2 = 0.99). Running average pO2 values (mean +/- SD) were also calculated for every minute and stored. The captured data files can be recalled and analyzed after completion of the perfusion experiment and compared with other data collected during perfusion. One of the unique capabilities of the pO2 electrodes is their ability to respond rapidly to changing perfusion conditions. For example, as the fetal circulation begins to break down the change is noted before volume loss in the fetal circuit by decreasing fetal vein pO2.