Assessment of gold nanoparticle effect on prostate cancer LNCaP cells

In recent years gold nanoparticles (AuNPs) have received considerable attention for various biomedical applications including diagnostics and targeted drug delivery. However, more research is still needed to characterize such aspects of their use in clinical oncology as permeability, retention and functional effect on tumor cells. Aims: This study was designed to describe the effect of non-functionalized AuNPs on LNCaP prostate cancer cells growth. Material and Methods: LNCaP cells were cultured in ­RPMI-1640 medium containing AuNPs covered by polyvinylpyrrolidone of average size 26.4 nm (10.0 μg/ml). Counts of cells were calculated and their morphology was examined. Results: AuNPs conglomerates have been visualized in cultured cells. After 4-day incubation in presence of AuNPs significant retardation of LNCaP cells growth was observed both in 5α-dihydrotestosterone stimulated and non-stimulated cultures. No morphological changes of live LNCaP cells were seen in any experiment. Conclusion: Given absence of morphological changes in live cells and dribble and relatively constant numbers of dead cells, it was concluded that inhibitory effect of AuNPs on LNCaP cells growth was caused by alterations of proliferation. Key Words: prostate cancer, LNCaP, gold nanoparticles, 5α-dihydrotestosterone

Diversity in protein–protein interactions of connexins: emerging roles

AbstractGap junctions, specialised membrane structures that mediate cell-to-cell communication in almost all tissues, are composed of channel-forming integral membrane proteins termed connexins. The activity of these intercellular channels is closely regulated, particularly by intramolecular modifications as phosphorylations of proteins by protein kinases, which appear to regulate the gap junction at several levels, including assembly of channels in the plasma membrane, connexin turnover as well as directly affecting the opening and closure (“gating”) of channels. The regulation of membrane channels by protein phosphorylation/dephosphorylation processes commonly requires the formation of a multiprotein complex, where pore-forming subunits bind to auxiliary proteins (e.g. scaffolding proteins, catalytic and regulatory subunits), that play essential roles in channel localisation and activity, linking signalling enzymes, substrates and effectors into a structure frequently anchored to the cytoskeleton. The present review summarises the up-to-date progress regarding the proteins capable of interacting or at least of co-localising with connexins and their functional importance

Temperature-dependent contractility of rat tunica dartos muscle: Contribution of cold, menthol-sensitive TRPM8

Tunica dartos smooth muscle (TDSM) lies beneath the scrotal skin, and its contraction leads to scrotum wrinkling upon cooling. However, neither the nature of TDSM cold-sensitivity nor the underlying molecular sensors are well understood. Here we have investigated the role of cold/menthol-sensitive TRPM8 channel in TDSM temperature-dependent contractility. The contraction of isolated male rat TDSM strips was studied by tensiometry. TRPM8 expression was assayed by RT-PCR and fluorescence immunochemistry. Isolated TDSM strips responded to cooling from 33 °C to 20 °C by enhancement of basal tension, and increase of the amplitude and duration of electric field stimulated (EFS) contractions. The effects of cold on basal tension, but not on EFS-contractions, could be 80% inhibited by TRPM8 blockers, capsazepine and BCTC [N-(4‑tert-butylphenyl)-4-(3-chloropyridin-2-yl)piperazine-1-carboxamide], and could be partially mimicked by menthol. RT-PCR and immunolabeling showed TRPM8 mRNA and protein expression in TDSM cells with protein labelling being predominantly localized to intracellular compartments. Chemical castration of male rats consequent to the treatment with androgen receptor blocker, flutamide, led to the abrogation of cold effects on TDSM basal tension, but not on EFS-contractions, and to the disappearance of TRPM8 protein expression. We conclude that TRPM8 is involved in the maintenance of basal cold-induced TDSM tonus, but not in sympathetic nerve-mediated contractility, by acting as endoplasmic reticulum Ca2+ release channel whose expression in TDSM cells requires the presence of a functional androgen receptor. Thus, TRPM8 plays a crucial role in scrotal thermoregulation which is important for maintaining normal spermatogenesis and male fertility

Calmodulin Mediates the Ca2+-Dependent Regulation of Cx44 Gap Junctions

We have shown previously that the Ca2+-dependent inhibition of lens epithelial cell-to-cell communication is mediated in part by the direct association of calmodulin (CaM) with connexin43 (Cx43), the major connexin in these cells. We now show that elevation of [Ca2+]i in HeLa cells transfected with the lens fiber cell gap junction protein sheep Cx44 also results in the inhibition of cell-to-cell dye transfer. A peptide comprising the putative CaM binding domain (aa 129–150) of the intracellular loop region of this connexin exhibited a high affinity, stoichiometric interaction with Ca2+-CaM. NMR studies indicate that the binding of Cx44 peptide to CaM reflects a classical embracing mode of interaction. The interaction is an exothermic event that is both enthalpically and entropically driven in which electrostatic interactions play an important role. The binding of the Cx44 peptide to CaM increases the CaM intradomain cooperativity and enhances the Ca2+-binding affinities of the C-domain of CaM more than twofold by slowing the rate of Ca2+ release from the complex. Our data suggest a common mechanism by which the Ca2+-dependent inhibition of the α-class of gap junction proteins is mediated by the direct association of an intracellular loop region of these proteins with Ca2+-CaM