Ion transport (36Cl uptake) and immunochemical studies were undertaken to detect the cystic fibrosis transmembrane conductance regulator (CFTR) in apical membrane vesicles prepared from human placenta. 36Cl uptake into membrane vesicles was studied in the absence and presence of inwardly directed potassium gradients and valinomycin (Ko = Ki and Ko > Ki, where Ko is potassium concentration outside and Ki is potassium concentration inside the vesicles). The sensitivities of 36Cl uptake to the inhibitors 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (DIDS), bumetanide, and diphenylamine-2-carboxylate were investigated. Each compound significantly inhibited uptake under both sets of conditions. Additional inhibition of 36Cl uptake was found when the compounds were added together, indicating that they were acting at least partly on different components of the 36Cl uptake. The DIDS- and bumetanide-insensitive component of transport was more selective for Cl than I. These findings suggested that this component may, at least in part, represent Cl transport via CFTR. Addition of adenosine 5'-O-(3-thiotriphosphate) (0.8 mM) led to a decrease in total 36Cl uptake but masked in the overall decrease was an increase in the DIDS- and bumetanide-insensitive component of 36Cl uptake. Western blot analysis of the apical membrane proteins with an antibody specific for a region of human CFTR detected a protein band of approximately 190 kDa. These ion transport and immunochemical studies provide evidence that CFTR is located in human placental apical membrane vesicles.

Apical membrane vesicles from human term placenta were isolated using a magnesium precipitation technique, and the purity of the vesicles was assessed morphologically using scanning and transmission electron microscopy, and biochemically, using marker enzymes. The vesicles were found to be morphologically intact and significantly enriched in enzymes associated with apical membranes. 36Cl- uptake into these vesicles was studied in the presence of an outwardly directed Cl- gradient. This uptake was found to be time dependent, with an initial rapid uptake tending to peak between 10 and 20 min and thereafter decline. Uptake was found to be voltage dependent since 5 microM valinomycin caused a decrease in uptake. The effects of N-phenylanthranilic acid (NPA) and 4,4'-diisothiocyanostilbene-2,2'-disulphonic acid (DIDS) and bumetanide on the initial rate of Cl- were examined in the presence and absence of 5 microM valinomycin. NPA and DIDS inhibited isotope uptake strongly with IC50 values of 0.83 +/- 0.35 microM and 3.43 +/- 0.37 microM, respectively, in the absence of valinomycin. Although valinomycin reduced 36Cl- uptake by about 80% when added before the isotope, DIDS reduced the uptake which remained in a concentration-dependent fashion with an IC50 of 5.6 +/- 2.1 microM. Under these conditions, NPA was without effect at concentrations below 100 microM. Bumetanide was without effect at the concentrations used in the absence of valinomycin. However, following valinomycin pretreatment, bumetanide reduced 36Cl- uptake significantly at 100 microM concentration. Vesicle diameter, as assessed by flow cytometry, did not change under the conditions employed. The effects of some fatty acids were also investigated. Arachidonic acid and linoleic acid inhibited Cl- uptake with IC50 values of 37.6 +/- 14.9 microM and 4.59 +/- 0.51 microM, respectively. Arachidonyl alcohol and elaidic acid were found to be without effect. These studies show that human placental brush border membrane vesicles possess a chloride conductance channel, the activity of which can be measured in the presence of an outwardly directed Cl- gradient and this channel is sensitive to Cl- channel inhibitors, especially N-phenylanthranilic acid, and can be inhibited by unsaturated fatty acids such as arachidonic acid and linoleic acid.

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Background/Aim: Isothiocyanates (ITCs) are phytochemicals with potential cancer-preventative properties derived from the breakdown of glucosinolates that exist in cruciferous vegetables. Studies, to date, have demonstrated that various ITCs possess the ability to act as anticancer agents in different cancer types. This study investigated the anticancer properties of dietary ITCs (allyl-ITC, benzyl-ITC, phenylethyl-ITC) and synthetic (phenylbutyl-ITC and phenylhexyl-ITC) on liver and prostate carcinoma cells in vitro. Materials and Methods: The effects of ITCs on cellular viability, migration, invasion, clonogenicity, apoptosis induction and reactive oxygen species generation were assessed in HepG2, DU145 and 22Rv1 cells. Results: All ITCs reduced metabolic activity in each cell line with the most significant being phenylethyl-ITC. Both dietary and synthetic ITCs suppressed the migratory and invasive potential of all cell lines, inhibited colony-forming capability and induced apoptosis. Phenylethyl-ITC exposure resulted in the significant generation of reactive oxygen species. Conclusion: These data highlight the potential advantages of utilizing ITCs to delay the carcinogenic process and the potential for dietary and synthetic ITCs to act as anticancer agents.

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