Caveolae-dependent Endocytosis Research Articles

Insights into the intracellular behaviors of black-phosphorus-based nanocomposites via surface-enhanced Raman spectroscopy

Abstract As one of the prospective two-dimensional nanomaterials, black phosphorus (BP), which has excellent physical and chemical properties, has witnessed quick development in theranostic applications. The more recent advances in combining BP nanosheet (NS) with nanoparticles exhibit new opportunities to develop multifunctional nanocomposites. However, more effort should be devoted to elucidate the nanomaterial-cell interaction mechanism before the bio-applications of BP-nanoparticle hybrids. Herein, the intracellular behaviors of BP-gold nanoparticles (BP-Au NSs) are first investigated using the surface-enhanced Raman scattering (SERS) technique. The presence of Au nanoparticles on the surface of a BP sheet allows nanohybrids with excellent SERS activity to enhance the intrinsic Raman signals of cellular components located around the NSs. Data from an endocytosis inhibitor blocking assay reveal that the nanohybrids are mainly taken up by macropinocytosis and caveolae-dependent endocytosis, which are energy-dependent processes. Associated with colocalization experiments, nanohybrids are found to internalize into lysosomes and the endoplasmic reticulum. Moreover, the SERS difference spectrum is extracted after Raman-fluorescence colocalization statistical analysis to distinguish the molecular structural differences in the biochemical components of the two organelles. These findings supply a definite cellular mechanistic understanding of the nano-biointeractions of nanocomposites in cancer cells, which may be of great importance to the biomedical applications of nanotechnology in the future.

Intracellular pathway of halloysite nanotubes: potential application for antitumor drug delivery

Natural halloysite nanotubes (HNTs), with nanotubular structure, are attracting considerable attention in recent years. The hollow tubular structure allows HNTs to play an important role in drug delivery system as drug carriers. However, the wide applications of HNTs in biomedicine have been hampered by the lack of sufficient intracellular researches so far. In this study, we systemically investigated the transport mechanisms of HNTs in A549 living cells. The colocalization and inhibition experiments illustrated FITC-labeled HNTs were readily internalized into cells by both clathrin- and caveolae-dependent endocytosis, and the transport pathway of HNTs is an actin- and microtubule-associated process via Golgi apparatus and lysosome. Meanwhile, the cell cycle assay clarified that HNTs can prompt the intracellular transportation of gemcitabine and enhance the gemcitabine concentration in A549 tumor cells. Such elucidation of intracellular transport pathway of HNTs offers insights into the site-specific delivery and cellular internalization of HNTs, which provide a reasonable guidance for the design of novel drug delivery system.

Mechanism of recipient cell-dependent differences in exosome uptake

BackgroundExosomes, small-membrane vesicles, are secreted by cells and include several types of proteins and nucleic acids. Exosomes transfer cellular information derived from donor cells and are involved in various physiological and pathological events, such as organ-specific metastasis. Elucidating the exosome uptake mechanisms is important for understanding the progression processes of organ-specific metastasis. However, whether the exosomes secreted by the donor cells are selectively or non-selectively incorporated into the recipient cells is unknown.MethodsIn this study, three human carcinoma cell lines, A549 (lung), HCT116 and COLO205 (colon), were used. The exosome isolation efficiency was compared between three methods: ultracentrifugation, ExoQuick-TC and Total Exosome Isolation kits. Recipient cells were treated with Pitstop 2, an inhibitor of clathrin-dependent endocytosis, or genistein, an inhibitor of caveolae-dependent endocytosis, and then incubated with DiO-labeled exosomes.ResultsAmong the three methods examined, ultracentrifugation was the most efficient and reproducible. Exosomes derived from a donor cell line are incorporated into the three cell lines, but the exosome uptake capability was different depending on the recipient cell type and did not depend on the donor cell type. Exosome uptake in COLO205 was inhibited by Pitstop 2 and genistein. Exosome uptake in HCT116 was inhibited by Pitstop 2, but not genistein, while that in A549 cells was not inhibited by these inhibitors. Taken together, these results suggest that the exosomes secreted by donor cells are non-selectively incorporated into recipient cells and that the exosome uptake mechanism is different depending on the recipient cells.ConclusionsDifferent recipient cells’ exosome uptake capabilities may be involved in organ-specific metastasis.

Gold nanoconjugates reinforce the potency of conjugated cisplatin and doxorubicin

Osteosarcoma or osteogenic sarcoma is the most common and prevalent cancerous tumor of bone and occurs especially in children and teens. Recent treatment strategy includes a combination of both chemotherapy and surgeries. Although, the use of single drug-based chemotherapy treatment remains unsatisfactory. Therefore, combinatorial therapy has emerged as a potential strategy for treatment with limited side- effects. Here, we evaluated the combinatorial anticancerous effect of cisplatin (CIS) and doxorubicin (DOX) bioconjugated bromelain encapsulated gold nanoparticles (B-AuNPs conjugated CIS and DOX) in the treatment of osteosarcoma. The synthesized B-AuNPs conjugated CIS and DOX were characterized by various characterization techniques like UV–vis spectroscopy, TEM, DLS and zeta potential to ensure the synthesis, size, shape, size distribution and stability. Drug loading efficiency bioconjugation of CIS and DOX was ensured by UV–vis spectroscopy. Bioconjugation of CIS and DOX was further confirmed using UV–vis spectroscopy, TEM, DLS, Zeta potential and FT-IR analysis. The combinatorial effect of CIS and DOX in B-AuNPs conjugated CIS and DOX showed highly improved potency against MG-63 and Saos-2 cells at a very low concentration where primary osteoblasts didn’t show any cytotoxic effect. The apoptotic effect of B-AuNPs conjugated CIS and DOX on osteosarcoma and primary osteoblasts cells were analyzed by increased permeability of the cell membrane, condensed chromatin and deep blue fluorescent condensed nucleus. The results clearly showed that B-AuNPs conjugated CIS and DOX significantly improved the potency of both the chemotherapeutic drugs by delivering them specifically into the nucleus of cancer cells through caveolae-dependent endocytosis. Thus, the greater inhibitory effect of combinatorial drugs (B-AuNPs conjugated CIS and DOX) over single drug based chemotherapy would be of great advantage during osteosarcoma treatment.

RGD-Modified Albumin Nanoconjugates for Targeted Delivery of a Porphyrin Photosensitizer.

Advances in photodynamic therapy of cancer have been restrained by lack of cancer specificity and side effects to normal tissues. Molecularly targeted photodynamic therapy can achieve higher cancer specificity by combination of active cancer targeting and localized laser activation. We aimed to use albumin as a carrier to prepare targeted nanoconjugates that are selective to cancer cells and smaller than conventional nanoparticles for superior tumor penetration. IRDye 700DX (IR700), a porphyrin photosensitizer, was covalently conjugated to human serum albumin that was also linked with tumor-targeting RGD peptides. With multiple IR700 and RGD molecules in a single albumin molecule, the resultant nanoconjugates demonstrated monodispersed and uniform size distribution with a diameter of 10.9 nm. These targeted nanoconjugates showed 121-fold increase in cellular delivery of IR700 into TOV21G ovarian cancer cells compared to control nanoconjugates. Mechanistic studies revealed that the integrin specific cellular delivery was achieved through dynamin-mediated caveolae-dependent endocytosis pathways. They produced massive cell killing in TOV21G cells at low nanomolar concentrations upon light irradiation, while NIH/3T3 cells that do not express integrin αvβ3 were not affected. Because of their small size, targeted albumin nanoconjugates could penetrate tumor spheroids of SKOV-3 ovarian cancer cells and produced strong phototoxicity in this 3-D model. Owing to their cancer-specific delivery and small size, these targeted nanoconjugates may become an effective drug delivery system for enabling molecularly targeted photodynamic therapy of cancer.

Abstract 5805: Myosin 1e colocalization with β1-intergin and association with tumor progression in colorectal cancer

Abstract For colorectal cancer (CRC), the risk of relapse among stage II and stage III a/b patients is 20-30% and the benefit of treating these patients with chemotherapy is uncertain. As a result, many patients are undertreated, putting them at increased risk for disease relapse, or overtreated, exposing them to unnecessary and harmful chemotherapy with little potential benefit. Therefore, defining prognostic biomarkers to more accurately determine each person’s risk of relapse and need for chemotherapy is a priority in CRC research. Promising targets for biomarker discovery are actin-binding proteins. As a broad class, these proteins regulate the actin cytoskeleton and serve as direct, proximal regulators of invasive and metastatic phenotypes. Further, the prognostic utility of several of these proteins has recently been reported in the literature. Myosin 1e (Myo1e) a long-tailed, class I myosin, is one such protein with proposed clinical utility as a prognostic biomarker. In kidney podocyte cells, Myo1e regulates endocytosis, adhesion, migration, and invadosome dynamics. Despite its expression in numerous cancers, the functional role of Myo1e specifically in cancer cells and its association with tumor progression remains elusive. To define a role in cancer cells, the localization of Myo1e was examined in vitro by immunofluorescence in T84 human CRC cells. Myo1e co-localized with actin, cortactin and β1 integrin at membrane ruffles, which regulate integrin endocytosis and trafficking. Interestingly, both Myo1e and β1 integrin also co-localized with caveolin-1, a regulator of integrin endocytosis, suggesting Myo1e expression may regulate caveolae-dependent integrin endocytosis and trafficking. To evaluate potential prognostic utility, Myo1e expression in colorectal tumors and matched normal adjacent tissue (NAT) was examined by immunohistochemistry in a tissue microarray constructed from duplicate tissue cores from 119 CRC patients. The patient cohort was well balanced across TNM stage: stage 0 (10.1%), stage 1 (20.2%), stage 2 (26.9%), stage 3 (31.1%), and stage 4 (12.6%), and the majority (68.9%) of CRC tissues examined were of moderate tumor grade. The ratio of Myo1e expression in tumors compared to NAT was significantly correlated with clinicopathologic indicators of disease progression, including clinical stage, depth of invasion at the primary tumor (T-score), and lymph node metastasis. Together, these data suggest Myo1e expression correlates with tumor progression and may regulate invasive phenotypes through integrin trafficking pathways. Examining the prognostic utility and biologic function of Myo1e may a define a novel biomarker with translational utility for improved clinical management of colorectal cancer patients. Citation Format: Jeffrey Pfannenstein, Filippo Bori, Alessandro Bombonati, David Zuzga. Myosin 1e colocalization with β1-intergin and association with tumor progression in colorectal cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5805. doi:10.1158/1538-7445.AM2017-5805

Cellular uptake mechanism and clearance kinetics of fluorescence-labeled glycyrrhetinic acid and glycyrrhetinic acid–modified liposome in hepatocellular carcinoma cells

Glycyrrhetinic acid (GA) is a natural pentacyclic triterpene derivative that exerts significant effects in the suppression of liver cancer. The receptors of GA on liver cells and hepatocellular carcinoma (HCC) cells have drawn broad attention. The effects of GA might depend on its transport into and out of cells. However, the question has not been previously addressed despite its obvious and fundamental importance. In this paper, GA and GA-modified liposome (GA-Lip) were labeled with fluorescein isothiocyanate (FITC) or coumarin 6 (Cou6) using chemical or pharmaceutical techniques. The transport courses of FITC-GA and GA-Cou6-Lip were studied in HepG2 cells in vitro. We found that the fluorescence labeled GA and GA-Lip uptake and clearance were time-dependent. FITC-GA uptake involved passive diffusion and active transport, and the receptors were in the cytomembrane proteins. GA-Cou6-Lip uptake was mediated by caveolae-dependent endocytosis. In addition, FITC-GA and GA-Cou6-Lip clearance of the HCC cells fitted exponential decay and second-order processes, respectively. These findings provide new insights into the anti–HCC actions of GA.

In vitro cellular behaviors and toxicity assays of small-sized fluorescent silicon nanoparticles.

Extensive investigations have been carried out for evaluating the toxicology of various nanomaterials (e.g., carbon- and metal-based nanomaterials), which offer invaluable information for assessing the feasibility of nanomaterial-based wide-ranging applications. In recent years, sufficient efforts have been made to develop fluorescent small-sized silicon nanoparticles (SiNPs) as a novel optical material simultaneously featuring strong fluorescence and ultrahigh photostability, providing high promise for a myriad of biological, biomedical and electronic applications. It is worth pointing out that, despite the non- or low-toxicity of silicon, sufficient and objective toxicology evaluation of SiNPs is urgently required at both the in vitro and in vivo levels. However, there currently exists scanty information about the intracellular behaviors of the SiNPs, particularly the underlying mechanism of entry into cells and intracellular fate. Herein, we present a report aimed at determining the uptake and intracellular transport of SiNPs of ca. 4 nm diameter. Taking advantage of the strong and stable fluorescent signals of SiNPs, we reveal that these small-sized SiNPs accumulate in the plasma membrane prior to internalization, and are further internalized predominantly by clathrin-mediated and caveolae-dependent endocytosis. After endocytosis, the SiNPs are localized in early endosomes within a short time (∼1 h), while in up to 24 h of incubation the SiNPs are mainly transported to lysosomes in a microtubule-dependent way; and interestingly, to a smaller extent are sorted to the Golgi apparatus. Moreover, we demonstrate that there are no toxic effects of SiNPs on the cell metabolic activity and integrity of the plasma membrane.

Newcastle disease virus employs macropinocytosis and Rab5a-dependent intracellular trafficking to infect DF-1 cells.

Oncolytic Newcastle disease virus (NDV) reportedly employs direct fusion of the viral envelope with the plasma membrane and caveolae-dependent endocytosis to enter cells. Here, we show that macropinocytosis and clathrin-mediated endocytosis are involved in NDV entry into a galline embryonic fibroblast cell line. Upon specific inhibition of clathrin assembly, GTPase dynamin, Na+/H+ exchangers, Ras-related C3 botulinum toxin substrate 1, p21 activated kinase 1 or protein kinase C, entry of NDV and its propagation were suppressed. NDV entry into cells triggers Rac1-Pak1 signaling and elicits actin rearrangement and plasma membrane ruffling. Moreover, NDV internalization within macropinosomes and trafficking involve Rab5a-positive vesicles. This is the first report demonstrating that NDV utilizes clathrin-mediated endocytosis and macropinocytosis as alternative endocytic pathways to enter cells. These findings shed new light on the molecular mechanisms underlying NDV entry into cells, and provide potential targets for NDV-mediated therapy in cancer.

Indirect effects of TiO2 nanoparticle on neuron-glial cell interactions

Although, titanium dioxide nanoparticles (TiO2NPs) are nanomaterials commonly used in consumer products, little is known about their hazardous effects, especially on central nervous systems. To examine this issue, ALT astrocyte-like, BV-2 microglia and differentiated N2a neuroblastoma cells were exposed to 6 nm of 100% anatase TiO2NPs. A lipopolysaccharide (LPS) was pre-treated to activate glial cells before NP treatment for mimicking NP exposure under brain injury. We found that ALT and BV-2 cells took up more NPs than N2a cells and caused lower cell viability. TiO2NPs induced IL-1β in the three cell lines and IL-6 in N2a. LPS-activated BV-2 took up more TiO2NPs than normal BV-2 and released more intra/extracellular reactive oxygen species (ROS), IL-1β, IL-6 and MCP-1 than did activated BV-2. Involvement of clathrin- and caveolae-dependent endocytosis in ALT and clathrin-dependent endocytosis and phagocytosis in BV-2 both had a slow NP translocation rate to lysosome, which may cause slow ROS production (after 24 h). Although TiO2NPs did not directly cause N2a viability loss, by indirect NP exposure to the bottom chamber of LPS-activated BV-2 in the Transwell system, they caused late apoptosis and loss of cell viability in the upper N2a chamber due to H2O2 and/or TNF-α release from BV-2. However, none of the adverse effects in N2a or BV-2 cells was observed when TiO2NPs were exposed to ALT-N2a or ALT-BV-2 co-culture. These results demonstrate that neuron damage can result from TiO2NP-mediated ROS and/or cytokines release from microglia, but not from astrocytes.

Insulin receptor trafficking steers insulin action.

Insulin receptor trafficking steers insulin action.

Dynamic bio-adhesion of polymer nanoparticles on MDCK epithelial cells and its impact on bio-membranes, endocytosis and paracytosis.

Nowadays, concern about the use of nanotechnology for biomedical application is unprecedentedly increasing. In fact, nanosystems applied for various potential clinical uses always have to cross the primary biological barrier consisting of epithelial cells. However, little is really known currently in terms of the influence of the dynamic bio-adhesion of nanosystems on bio-membranes as well as on endocytosis and transcytosis. This was investigated here using polymer nanoparticles (PNs) and MDCK epithelial cells as the models. Firstly, the adhesion of PNs on cell membranes was found to be time-dependent with a shift of both location and dispersion pattern, from the lateral adhesion of mainly mono-dispersed PNs initially to the apical coverage of the PN aggregate later. Then, it was interesting to observe in this study that the dynamic bio-adhesion of PNs only affected their endocytosis but not their transcytosis. It was important to find that the endocytosis of PNs was not a constant process. A GM1 dependent CDE (caveolae dependent endocytosis) pathway was dominant in the preliminary stage, followed by the co-existence of a CME (clathrin-mediated endocytosis) pathway for the PN aggregate at a later stage, in accordance with the adhesion features of PNs, suggesting the modification of PN adhesion patterns on the endocytosis pathways. Next, the PN adhesion was noticed to affect the structure of cell junctions, via altering the extra- and intra-cellular calcium levels, leading to the enhanced paracellular transport of small molecules, but not favorably enough for the obviously increased passing of PNs themselves. Finally, FRAP and other techniques all demonstrated the obvious impact of PN adhesion on the membrane confirmation, independent of the adhesion location and time, which might lower the threshold for the internalization of PNs, even their aggregates. Generally, these findings confirm that the transport pathway mechanism of PNs through epithelial cells is rather dynamic, and is remarkably affected by the adhesion patterns of PNs on the cell membrane.

Role of endocytosis in sonoporation-mediated membrane permeabilization and uptake of small molecules: a electron microscopy study

Sonoporation is a physical method that has been successfully used to deliver drugs into living cells both in vitro and in vivo for experimental and therapeutic purposes. Despite numerous studies on this topic, often reporting successful outcomes, very little is known about the mechanisms involved in the hypothesized membrane permeabilization processes. In this study, electron microscopy was used to investigate the ultra-structural modifications of cell membranes, induced by sonoporation. Here, we demonstrate that sonoporation in the presence of microbubbles induces the formation of a significant number of transient and permeant structures at the membrane level. These structures were transient with a half-life of 10 min and had a heterogeneous size distribution ranging from a few nanometers to 150 nm. We demonstrated that the number and the size of these structures were positively correlated with the enhanced intracellular uptake of small molecules. In addition, we showed that these structures were associated with caveolae-dependent endocytosis for two thirds of the recorded events, with the remaining one third related to non-specific routes such as membrane disruptions as well as caveolae-independent endocytosis. In conclusion, our observations provide direct evidences of the involvement of caveolae-endocytosis in cell membrane permeabilization to small molecules after sonoporation.

RGD functionalized polymeric nanoparticles targeting periodontitis epithelial cells for the enhanced treatment of periodontitis in dogs

Long term retention of antimicrobials with effective drug concentration in gingival crevicular fluid (GCF) is of vital importance for the treatment of chronic periodontitis. In this study, a novel epithelial cell-targeting nanoparticle drug delivery system by conjugating minocycline-loaded poly(ethylene glycol)–poly(lactic acid) (PEG–PLA) nanoparticles (NP-MIN) with RGD peptide were developed and administrated locally for targeting periodontitis epithelial cells and enhancing the treatment of periodontitis in dogs. Biodegradable NP-MIN was made with an emulsion/solvent evaporation technique. RGD peptide was conjugated to the surface of nanoparticles via Maleimide group reaction with hydrosulfide in RGD peptide (RGD-NP-MIN). Transmission electron microscopy examination and dynamic light scattering results revealed that RGD-NP-MIN had a sphere shape, with a mean diameter around 106nm. In vitro release of minocycline from RGD-NP-MIN showed that RGD modification did not change the remarkable sustained releasing characteristic of NP-MIN. To elucidate the interaction of RGD-NP and epithelial cells, RGD-NP binding, uptake and cellular internalization mechanisms by calu-3 cells were investigated. It was shown RGD modification significantly enhanced nanoparticles binding and uptake by Calu-3 cells, and RGD-NP uptake was an energy-dependent process through receptor-mediated endocytosis. Both clathrin-associated endocytosis and caveolae-dependent endocytosis pathway were involved in the RGD-NP uptake, and the intracellular transport of RGD-NP was related to lysosome and Golgi apparatus. Finally, in vivo pharmacokinetics of minocycline in the periodontal pockets and anti-periodontitis effects of RGD-NP-MIN on periodontitis-bearing dogs were evaluated. After local administration of RGD-NP-MIN, minocycline concentration in gingival crevicular fluid decreased slowly and maintained an effective drug concentration for a longer time than that of NP-MIN. Anti-periodontitis effects demonstrated that RGD-NP-MIN could significantly decrease symptoms of periodontitis, which was better than any other control group. These findings suggested that these epithelial cell-targeting nanoparticles offered a novel and effective local delivery system for the treatment of periodontitis.

Real-time Imaging of Rabies Virus Entry into Living Vero cells.

Understanding the mechanism of rabies virus (RABV) infection is vital for prevention and therapy of virulent rabies. However, the infection mechanism remains largely uncharacterized due to the limited methods and viral models. Herein, we utilized a powerful single-virus tracking technique to dynamically and globally visualize the infection process of the live attenuated rabies vaccine strain-SRV9 in living Vero cells. Firstly, it was found that the actin-enriched filopodia is in favor of virus reaching to the cell body. Furthermore, by carrying out drug perturbation experiments, we confirmed that RABV internalization into Vero cells proceeds via classical dynamin-dependent clathrin-mediated endocytosis with requirement for intact actin, but caveolae-dependent endocytosis is not involved. Then, our real-time imaging results unambiguously uncover the characteristics of viral internalization and cellular transport dynamics. In addition, our results directly and quantitatively reveal that the intracellular motility of internalized RABV particles is largely microtubule-dependent. Collectively, our work is crucial for understanding the initial steps of RABV infection, and elucidating the mechanisms of post-infection. Significantly, the results provide profound insight into development of novel and effective antiviral targets.

Hypergravity-induced enrichment of β1 integrin on the cell membranes of osteoblast-like cells via caveolae-dependent endocytosis

In bone cells, integrins on the cellular surface are the primary sensors of their mechanical environment. Although gravitational changes are known to affect the adhesion and functions of bone cells, whether integrins respond to hypergravity in osteoblasts remains unclear. In this work, we demonstrate that exposure to a hypergravitational environment (20 × g via centrifugation) resulted in the concentration of β1, but not β3, integrin on the cell membrane of osteoblast-like (MC3T3-E1) cells. Notably, the total expression of both integrins was unaffected by the hypergravitational environment. In addition, caveolin-dependent endocytosis was discovered to be involved in the regulation of the enrichment of β1 integrin on the cell surface after stimulation by hypergravity. These findings could aid in the improvement of our understanding of the mechanisms underlying the effects of different gravitational forces on the human body.

Selectivity of commonly used inhibitors of clathrin-mediated and caveolae-dependent endocytosis of G protein–coupled receptors

Among the multiple G protein–coupled receptor (GPCR) endocytic pathways, clathrin-mediated endocytosis (CME) and caveolar endocytosis are more extensively characterized than other endocytic pathways. A number of endocytic inhibitors have been used to block CME; however, systemic studies to determine the selectivity of these inhibitors are needed. Clathrin heavy chain or caveolin1-knockdown cells have been employed to determine the specificity of various chemical and molecular biological tools for CME and caveolar endocytosis. Sucrose, concanavalin A, and dominant negative mutants of dynamin blocked other endocytic pathways, in addition to CME. In particular, concanavalin A nonspecifically interfered with the signaling of several GPCRs tested in the study. Decreased pH, monodansylcadaverine, and dominant negative mutants of epsin were more specific for CME than other treatments were. A recently introduced CME inhibitor, Pitstop2™, showed only marginal selectivity for CME and interfered with receptor expression on the cell surface. Blockade of receptor endocytosis by epsin mutants and knockdown of the clathrin heavy chain enhanced the β2AR-mediated ERK activation. Overall, our studies show that previous experimental results should be interpreted with discretion if they included the use of endocytic inhibitors that were previously thought to be CME-selective. In addition, our study shows that endocytosis of β2 adrenoceptor through clathrin-mediated pathway has negative effects on ERK activation.

Phospholipid-stabilized mesoporous carbon nanospheres as versatile carriers for systemic delivery of amphiphobic SNX-2112 (a Hsp90 inhibitor) with enhanced antitumor effect

Systemic delivery of amphiphobic drugs (insoluble in both water and oil) represents a formidable challenge in drug delivery. This work aimed to engineer a functional mesoporous carbon material to efficiently load SNX-2112, an amphiphobic anticancer agent, and to evaluate its performance in tumor-targeting delivery. Hydrothermal reaction combined with high-temperature activation was used to fabricate glucose-based mesoporous carbon nanospheres (MCNs). SNX-2112-loaded MCNs stabilized by phospholipid (SN-PMCNs) were prepared by the absorption/solvent diffusion/high-pressure homogenization method. The obtained SN-PMCNs were 180nm around in particle size, showing a high drug load (42.7%) and acceptable physical stability. SN-PMCNs demonstrated an enhanced in vitro antitumor effect and increased uptake into cancer cells in comparison with the formulation of SNX-2112 solution (SN-Sol). The in vivo antitumor effect and biodistribution in 4T1 xenograft tumor mice, a breast cancer model, were also significantly improved through SN-PMCNs. It was shown that specific clathrin-dependent and nonspecific caveolae-dependent endocytosis were involved in the cellular trafficking of SN-PMCNs. Glucose transporter-mediated transport, prolonged body residence time and improved biodistribution via EPR effect were the main mechanisms of enhanced antitumor effect. SN-PMCNs have presented excellent tumor targeting properties and should be a promising carrier to address the systemic delivery of SNX-2112.

The Dioxin receptor modulates Caveolin-1 mobilization during directional migration: role of cholesterol.

BackgroundAdhesion and migration are relevant physiological functions that must be regulated by the cell under both normal and pathological conditions. The dioxin receptor (AhR) has emerged as a transcription factor regulating both processes in mesenchymal, epithelial and endothelial cells. Indirect results suggest that AhR could cooperate not only with additional transcription factors but also with membrane-associated proteins to drive such processes.ResultsIn this study, we have used immortalized and primary dermal fibroblasts from wild type (AhR+/+) and AhR-null (AhR−/−) mice to show that AhR modulates membrane distribution and mobilization of caveolin-1 (Cav-1) during directional cell migration. AhR co-immunoprecipitated with Cav-1 and a fraction of both proteins co-localized to detergent-resistant membrane microdomains (DRM). Consistent with a role of AhR in the process, AhR−/− cells had a significant reduction in Cav-1 in DRMs. Moreover, high cell density reduced AhR nuclear levels and moved Cav-1 from DRMs to the soluble membrane in AhR+/+ but not in AhR−/− cells. Tyrosine-14 phosphorylation had a complex role in the mechanism since its upregulation reduced Cav-1 in DRMs in both AhR+/+ and AhR−/−cells, despite the lower basal levels of Y14-Cav-1 in the null cells. Fluorescence recovery after photobleaching revealed that AhR knock-down blocked Cav-1 transport to the plasma membrane, a deficit possibly influencing its depleted levels in DRMs. Membrane distribution of Cav-1 in AhR-null fibroblasts correlated with higher levels of cholesterol and with disrupted membrane microdomains, whereas addition of exogenous cholesterol changed the Cav-1 distribution of AhR+/+ cells to the null phenotype. Consistently, higher cholesterol levels enhanced caveolae-dependent endocytosis in AhR-null cells.ConclusionsThese results suggest that AhR modulates Cav-1 distribution in migrating cells through the control of cholesterol-enriched membrane microdomains. Our study also supports the likely possibility of membrane-related, transcription factor independent, functions of AhR.Electronic supplementary materialThe online version of this article (doi:10.1186/s12964-014-0057-7) contains supplementary material, which is available to authorized users.

Compounds with anti-influenza activity: present and future of strategies for the optimal treatment and management of influenza. Part I: Influenza life-cycle and currently available drugs.

Influenza is a contagious respiratory acute viral disease characterized by a short incubation period, high fever and respiratory and systemic symptoms. The burden of influenza is very heavy. Indeed, the World Health Organization (WHO) estimates that annual epidemics affect 5-15% of the world's population, causing up to 4-5 million severe cases and from 250,000 to 500,000 deaths. In order to design anti-influenza molecules and compounds, it is important to understand the complex replication cycle of the influenza virus. Replication is achieved through various stages. First, the virus must engage the sialic acid receptors present on the free surface of the cells of the respiratory tract. The virus can then enter the cells by different routes (clathrin-mediated endocytosis or CME, caveolae-dependent endocytosis or CDE, clathrin-caveolae-independent endocytosis, or macropinocytosis). CME is the most usual pathway; the virus is internalized into an endosomal compartment, from which it must emerge in order to release its nucleic acid into the cytosol. The ribonucleoprotein must then reach the nucleus in order to begin the process of translation of its genes and to transcribe and replicate its nucleic acid. Subsequently, the RNA segments, surrounded by the nucleoproteins, must migrate to the cell membrane in order to enable viral assembly. Finally, the virus must be freed to invade other cells of the respiratory tract. All this is achieved through a synchronized action of molecules that perform multiple enzymatic and catalytic reactions, currently known only in part, and for which many inhibitory or competitive molecules have been studied. Some of these studies have led to the development of drugs that have been approved, such as Amantadine, Rimantadine, Oseltamivir, Zanamivir, Peramivir, Laninamivir, Ribavirin and Arbidol. This review focuses on the influenza life-cycle and on the currently available drugs, while potential antiviral compounds for the prevention and treatment of influenza are considered in the subsequent review.