Coupling of TRPV6 and TMEM16A in epithelial principal cells of the rat epididymis.

The epididymis establishes a congenial environment for sperm maturation and protection. Its fluid is acidic, and the calcium concentration is low and declines along the length of the epididymal tubule. However, our knowledge of ionic currents and mechanisms of calcium homeostasis in rat epididymal epithelial cells remains enigmatic. In this study, to better understand calcium regulation in the epididymis, we use the patch-clamp method to record from single rat cauda epididymal principal cells. We detect a constitutively active Ca(2+) current with characteristics that match the epithelial calcium channel TRPV6. Electrophysiological and pharmacological data also reveal a constitutively active calcium-activated chloride conductance (CaCC). Removal of extracellular calcium attenuates not only the TRPV6-like conductance, but also the CaCC. Lanthanide block is time dependent such that the TRPV6-like component is inhibited first, followed by the CaCC. The putative CaCC blocker niflumic acid partially inhibits whole-cell currents, whereas La(3+) almost abolishes whole-cell currents in principal cells. Membrane potential measurements reveal an interplay between La(3+)-sensitive ion channels and those that are sensitive to the specific TMEM16A inhibitor tannic acid. In vivo perfusion of the cauda epididymal tubule shows a substantial rate of Ca(2+) reabsorption from the luminal side, which is dose-dependently suppressed by ruthenium red, a putative blocker of epithelial Ca(2+) channels and CaCC. Finally, we discover messenger RNA for both TRPV6 and TMEM16A in the rat epididymis and show that their proteins colocalize in the apical membrane of principal cells. Collectively, these data provide evidence for a coupling mechanism between TRPV6 and TMEM16A in principal cells that may play an important role in the regulation of calcium homeostasis in the epididymis.

Proliferation and enrichment of CD133(+) glioblastoma cancer stem cells on 3D chitosan-alginate scaffolds.

Emerging evidence implicates cancer stem cells (CSCs) as primary determinants of the clinical behavior of human cancers, representing an ideal target for next-generation anti-cancer therapies. However CSCs are difficult to propagate in vitro, severely limiting the study of CSC biology and drug development. Here we report that growing cells from glioblastoma (GBM) cell lines on three dimensional (3D) porous chitosan-alginate (CA) scaffolds dramatically promotes the proliferation and enrichment of cells possessing the hallmarks of CSCs. CA scaffold-grown cells were found more tumorigenic in nude mouse xenografts than cells grown from monolayers. Growing in CA scaffolds rapidly promoted expression of genes involved in the epithelial-to-mesenchymal transition that has been implicated in the genesis of CSCs. Our results indicate that CA scaffolds have utility as a simple and inexpensive means to cultivate CSCs in vitro in support of studies to understand CSC biology and develop more effective anti-cancer therapies.

Aligned chitosan-polycaprolactone polyblend nanofibers promote the migration of glioblastoma cells.

In vitro models that accurately mimic the microenvironment of invading glioblastoma multiform (GBM) cells will provide a high-throughput system for testing potential anti-invasion therapies. Here, the ability of chitosan-polycaprolactone polyblend nanofibers to promote a migratory phenotype in human GBM cells by altering the nanotopography of the nanofiber membranes is investigated. Fibers are prepared with diameters of 200 nm, 400 nm, and 1.1 µm, and are either randomly oriented or aligned to produce six distinct nanotopographies. Human U-87 MG GBM cells, a model cell line commonly used for invasion assays, are cultured on the various nanofibrous substrates. Cells show elongation and alignment along the orientation of aligned fibers as early as 24 h and up to 120 h of culture. After 24 h of culture, human GBM cells cultured on aligned 200 nm and 400 nm fibers show marked upregulation of invasion-related genes including ß-catenin, Snail, STAT3, TGF-ß, and Twist, suggesting a mesenchymal change in these migrating cells. Additionally, cells cultured on 400 nm aligned fibers show similar migration profiles as those reported in vivo, and thus these nanofibers should provide a unique high-throughput in vitro culture substrate for developing anti-migration therapies for the treatment of GBM.

Chlorotoxin bound magnetic nanovector tailored for cancer cell targeting, imaging, and siRNA delivery.

Ribonucleic acid interference (RNAi) is a powerful molecular tool that has potential to revolutionize the treatment of cancer. One major challenge of applying this technology for clinical application is the lack of site-specific carriers that can effectively deliver short interfering RNA (siRNA) to cancer cells. Here we report the development and assessment of a cancer-cell specific magnetic nanovector construct for efficient siRNA delivery and non-invasive monitoring through magnetic resonance imaging (MRI). The base of the nanovector construct is comprised of a superparamagnetic iron oxide nanoparticle core coated with polyethylene glycol (PEG)-grafted chitosan, and polyethylenimine (PEI). The construct was then further functionalized with siRNA and a tumor-targeting peptide, chlorotoxin (CTX), to improve tumor specificity and potency. Flow cytometry, quantitative RT-PCR, and fluorescence microscopy analyses confirmed receptor-mediated cellular internalization of nanovectors and enhanced gene knockdown through targeted siRNA delivery. The ability of this nanovector construct to generate specific contrast enhancement of glioblastoma cells was demonstrated through MR imaging. These findings suggest that this CTX enabled nanoparticle carrier may be well suited for delivery of RNAi therapeutics to brain cancer cells.

Chitosan-alginate 3D scaffolds as a mimic of the glioma tumor microenvironment.

Despite recent advances in the understanding of its cell biology, glioma remains highly lethal. Development of effective therapies requires a cost-effective in vitro tumor model that more accurately resembles the in vivo tumor microenvironment as standard two-dimensional (2D) tissue culture conditions do so poorly. Here we report on the use of a three-dimensional (3D) chitosan-alginate (CA) scaffold to serve as an extracellular matrix that promotes the conversion of cultured cancer cells to a more malignant in vivo-like phenotype. Human U-87 MG and U-118 MG glioma cells and rat C6 glioma cells were chosen for the study. In vitro tumor cell proliferation and secretion of factors that promote tumor malignancy, including VEGF, MMP-2, fibronectin, and laminin, were assessed. The scaffolds pre-cultured with U-87 MG and C6 cells were then implanted into nude mice to evaluate tumor growth and blood vessel recruitment compared to the standard 2D cell culture and 3D Matrigel matrix xenograft controls. Our results indicate that while the behavior of C6 cells showed minimal differences due to their highly malignant and invasive nature, U-87 MG and U-118 MG cells exhibited notably higher malignancy when cultured in CA scaffolds. CA scaffolds provide a 3D microenvironment for glioma cells that is more representative of the in vivo tumor, thus can serve as a more effective platform for development and study of anticancer therapeutics. This unique CA scaffold platform may offer a valuable alternative strategy to the time-consuming and costly animal studies for a wide variety of experimental designs.

Cell-cell interaction underlies formation of fluid in the male reproductive tract of the rat.

The epithelia lining the epididymides of many species consists of several cell types. We have provided evidence that the basal cells are essential to the integrated functions of the epithelium. Basal cells, but not principal cells, and other cells in the epididymis express TRPC3 and COX-1. We have isolated basal cells from intact rat epididymis using antibody-coated Dynabeads and subjected them to whole-cell patch-clamp measurement of nonselective cation channel activity, a feature of TRPC3 protein, and Fluo-3 fluorescence measurement of intracellular Ca2+ concentration. The results show that a nonselective cation current blockable by La3+ (0.1 mM), Gd3+ (0.1 mM), or SKF96365 (20 microM) could be activated by lysylbradykinin (200 nM). In cells loaded with Fluo-3, addition of lysylbradykinin (100 nM) caused a sustained increase of intracellular Ca2+. This effect was blocked by Gd3+ (0.1 mM) or SKF96365 (20 microM) and was not observed in Fluo-3-loaded principal cells. Stimulation of basal cell/principal cell cocultures with lysylbradykinin (200 nM) evoked in principal cells a current with CFTR-Cl- channel characteristics. Isolated principal cells in the absence of basal cells did not respond to lysylbradykinin but responded to PGE2 (100 nM) with activation of a CFTR-like current. Basal cells, but not principal cells, released prostaglandin E2 when stimulated with lysylbradykinin (100 nM). The release was blocked by SKF96365 (20 microM) and BAPTA-AM (0.05 or 0.1 mM). Confluent cell monolayers harvested from a mixture of disaggregated principal cells and basal cells responded to lysylbradykinin (100 nM) and PGE2 (500 nM) with an increase in electrogenic anion secretion. The former response was dependent on prostaglandin synthesis as piroxicam blocked the response. However, cell cultures obtained from principal cells alone responded to PGE2 but not to bradykinin. These results support the notion that basal cells regulate principal cells through a Ca2+ and COX signaling pathway.