Significance Nontoxic agonists and antagonists of the aryl hydrocarbon receptor (AhR) hold high therapeutic potential for treating autoimmune disease and cancer. However, AhR activation by different ligands can lead to opposing phenotypical outcomes in a cell- and tissue-specific manner. In this study, we demonstrate that proportional flux in the levels of aryl hydrocarbon receptor nuclear translocator (ARNT) isoforms 1 and 3 modulates AhR signaling in terms of amplitude and expression of distinct gene programs. These results delineate a molecular mechanism of ARNT isoform–mediated AhR regulation, simplify our understanding of a complex AhR signaling pathway, and provide feasibility for ARNT-targeted therapies that could be used in conjunction with nontoxic AhR ligands for the purpose of immunomodulation.

Aims The purpose of this study was to determine the cell viability and cytotoxicity of various endocytosis and efflux inhibitors which can be used to determine transport and uptake mechanisms in the BeWo (b30 clone) human placental trophoblast cell line. Ethanol and dimethylsulfoxide (DMSO) were also studied since they are often used as cosolvents for administration of these inhibitors. Methodology The water-soluble tetrazolium-1 (WST-1) assay was used to quantify cell viability and the lactate dehydrogenase (LDH) assay was used to determine cytotoxicity. Results By the WST-1 assay, reduced cell viability was observed following 4 hours of exposure to chlorpromazine (10 μg/mL), colchicine (1 mM), filipin (3 μg/mL), gentamicin (2 mM), GF120918 (1 μM), methyl-β-cyclodextrin (5 mM), and verapamil (100 μM). By the LDH assay, however, no cytotoxicity was observed after 4 hours of exposure to the aforementioned compounds. Amiloride (500 μM), ethanol (up to 0.1% v/v), and DMSO (up to 0.1% v/v) did not reduce cell viability nor induce cytotoxicity. Conclusion This information is valuable when selecting potential inhibitors of endocytosis and efflux and the selection of time points for mechanistic studies.

Assessment of drug transport across the placenta is important in understanding the effect of drugs on placental and fetal health. These phenomena can be studied in both in vitro cell lines and ex vivo placental perfusions. We have successfully developed a sensitive yet simple high performance liquid chromatography (HPLC) method coupled with fluorescence detection to determine the concentration of doxorubicin (DXR) in cell culture media for transport studies in human trophoblast cells (BeWo, b30 clone) and in fetal media for placental perfusion experiments. The method was developed based on a protein precipitation technique and was validated in both media types for linearity, intra-day, and inter-day precision and accuracy. The relationship of peak area to concentration was linear with R2 values of 0.99 or greater obtained over the concentration range of 1.5 to 15,000 ng/mL. Despite the high concentrations of albumin in fetal perfusion media (30 mg/mL), the lower limits of detection and quantification for DXR were found to be 1.5 and 5 ng/mL, respectively. This analytical method may be used to study the transport of DXR across BeWo cells and human placenta during placental perfusion studies.

Background: Breast cancer affects 1 of every 3000 pregnancies, and is treated with chemotherapeutics including paclitaxel. Chemotherapy administration in pregnancy is associated with fetal morbidity. Purpose: Here, we investigated the transplacental transfer of both paclitaxel and new paclitaxel nanoformulations that are entering clinical studies or are commercially available for use in breast cancer. In addition, we examined the transfer of fluorescent albumin-based and polymeric micellar nanoformulations with similar properties to those paclitaxel nanoformulations. Methods: Transport studies were performed using BeWo b30 cells (human placental trophoblast cells) seeded in TranswellTM plates coated with human placental collagen. Cells were treated with 20 μg/mL of paclitaxel from marketed formulations. Since paclitaxel undergoes efflux by P-glycoprotein (P-gp) substrate, 100 μM of verapamil was used as a competitive P-gp inhibitor to prevent efflux of free paclitaxel released from the nanoformulations. In order to determine the role of nanomaterials in transport and uptake of paclitaxel, we synthesized fluorescent albumin nanoparticles and fluorescent polymeric micelles with similar properties to the paclitaxel nanoformulations for use in transport studies. Samples were analyzed by high-performance liquid chromatography or fluorometer. Uptake measurements were corrected for protein content. Particle size and polydispersity index were determined using dynamic light scattering. Results: There were no formulation-dependent differences in paclitaxel permeability between the formulations of paclitaxel, nanoparticle albumin-bound paclitaxel (nab-paclitaxel), and paclitaxel-loaded polymeric micelles. In the presence of verapamil, nab-paclitaxel showed a twofold increase in permeability, in contrast to the other formulations. The size of fluorescent albumin nanoparticles (130.3nm) was matched with nab-Paclitaxel (156.2 nm), and the fluorescent micelles (27.6 nm) with paclitaxel-loaded polymeric micelles (27.7nm). The apparent permeability of the fluorescent albumin nanoparticles across the BeWo cells was 1.98 x 10-6 ± 0.81 x 10-6 cm/s, whereas the apparent permeability of the fluorescent micelles was 59.5 x 10-6 ± 10.5 x 10-6 cm/s. Conclusion: These data suggest that paclitaxel-encapsulated micelles may evade efflux by P-gp and cross the trophoblast to a significant extent, while paclitaxel albumin nanoparticles behave like free paclitaxel. Therefore, choice of formulation may have a significant impact on fetal exposure to paclitaxel. We anticipate that these findings will have an impact on future pharmaceutical design and rational development of safe treatment strategies for pregnancy-associated breast cancer and other diseases. Citation Format: Shariq Ali, Sanaalarab Alenazy, Mansi Shah, Luke Bourner, Erik Rytting. Efflux of paclitaxel nanoformulation in cell culture model of human placental barrier [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2017 Oct 26-30; Philadelphia, PA. Philadelphia (PA): AACR; Mol Cancer Ther 2018;17(1 Suppl):Abstract nr A118.