Clathrin-mediated Endocytic Pathway Research Articles

Modulation of infectious Salmon Anaemia virus infection by clathrin-mediated endocytosis and macropinocytosis inhibitors

Infectious salmon anaemia virus (ISAV) is a pathogen that causes disease and large mortality in farm-raised Salmo salar L., being considered as a major problem in the salmon industry. However, despite its relevance, there are still numerous knowledge gaps on virus entry and early stages of infection. Previous studies suggested that virus entry into cells occurs via endocytosis, with no description of specific mechanisms. However, it remains unknown if the endocytosis induced by ISAV is a clathrin-dependent or clathrin-independent process. This study aimed to identify cellular mechanisms allowing ISAV entry into Atlantic Salmon head kidney (ASK) cells. Our results showed that ISAV can be found in coated pits and membrane ruffles, the latter being induced by a rearrangement of actin filaments promoted by ISAV infection. Additionally, it was determined that ISAV stimulate the uptake of extracellular fluid in a multiplicity of infection (MOI)-dependent manner. When the clathrin-mediated endocytic pathway was pharmacologically inhibited, ISAV infection was significantly reduced but not entirely inhibited. Similarly, when the Na+/H+ exchanger (NHE), a key component of macropinocytosis, was inhibited, ISAV infection was negatively affected. Our results suggest that ISAV enters cells via both clathrin-mediated endocytosis and most likely macropinocytosis.

Human ovarian granulosa cells use clathrin-mediated endocytosis for LDL uptake: immunocytochemical and electron microscopic study

ABSTRACT The steroidogenic activity of the granulosa cells is important for the reproductive cycle, and lipoproteins are involved in this process. The clathrin-mediated endocytosis pathway for LDL transport is considered to be the main one in eukaryotic cells. However, there are no studies that elucidate LDL internalization in human granulosa cells clarifying whether the clathrin-mediated endocytic pathway is functional in this process. The aim of this study is to investigate the role of clathrin and v-SNARE proteins in the formation of vesicles in human granulosa cells. In this study, the COV434 human granulosa cells were cultured and divided into four groups where in some of the groups Dil-conjugated LDL and Icarugamycin (ICA) a clathrin-mediated endocytosis inhibitor were added. From the collected mediums pregnenolone and progesterone levels were measured using ELISA. Oil red O staining was performed to show the intracellular lipids in the cells. Clathrin-coated vesicles believed to be responsible for carrying LDL, and v-SNARE proteins that direct the vesicles to their target molecules were also labeled and investigated by histological and ultrastructural methods. Our results show that human granulosa cells as well use the LDL cholesterol for steroid biosynthesis and they may prefer the clathrin-mediated endocytotic pathway to internalize it.

Advances in vesicle trafficking of membrane proteins and their regulatory mechanisms

<p indent="0mm">Membrane proteins are essential components of cell membrane structures and can be transported through vesicles as signaling molecules. The vesicle trafficking of membrane proteins is crucial for maintaining biological processes such as plant growth and development, substance exchange, cell recognition, immune response, and signal transduction. Both vesicle exocytosis and endocytosis play integral roles in regulating the activities and turnover of plasma membrane proteins required for signal triggering or attenuation at the cell surface. Exocytosis involves the transport of newly synthesized or recycled material to the plasma membrane (PM) in vesicles where they are released into the extracellular space. Recent investigations have shown that changing the balance between conventional and unconventional protein secretion can be an efficient strategy to respond to stressful conditions in eukaryotes. With the aid of an N-terminal located signal peptide (SP), conventional protein secretion delivers newly synthesized proteins from the endoplasmic reticulum (ER) to the Golgi apparatus and the <italic>trans</italic>-Golgi network (TGN) and finally to the PM or extracellular space via secretory vesicles or secretory granules. Unconventional protein secretion is responsible for the secretion of such SP-lacking protein. Notably, several unconventional protein secretion pathways mediated by distinct mechanisms have been reported in plants, most of which are related to stress responses. Conversely to exocytosis, endocytosis is the process by which material associated with the cellular surface is uptaken by invagination of the plasma membrane and internalized as an endocytic vesicle. In recent years, substantial progress has been made in the characterization of the plant components involved in clathrin-mediated and membrane microdomain-associated endocytic pathways. Moreover, the coordination of exocytic and endocytic trafficking has been shown to be important for various plant cell functions. An increasing pool of evidence has indicated that multiple cellular processes require tightly regulated exocytosis-endocytosis coupling, including nutrient uptake, signaling transduction, and plant–microbe interactions. However, compared to animal systems in which multiple mechanisms have been described, the functions and regulation of coupled trafficking processes in plants remain poorly understood. All proteins in the cells of an organism and most extracellular proteins are continually being degraded and replaced. The autophagy pathway is a conserved protein auto degradation pathway that delivers dysfunctional organelles or other cytoplasmic components to vacuoles for degradation and recycling in plants. In addition, autophagy is reported to be involved in plant development as part of processes such as root tip cell growth and differentiation. Recent observations have revealed possible interplay between vesicle trafficking and autophagic pathways in regulating vacuolar degradation in plants. In recent years, with constant advances in super-resolution microscopy and labeling methods, much progress has been made in mechanistic studies of membrane protein transportation, and a diversity of exocytic and endocytic trafficking pathways have been uncovered in plants. Although a variety of methods and techniques have been used to study plant vesicle trafficking, a systematic summary of vesicle trafficking pathways of plant membrane proteins has not been published. This review first introduces the relevant organelles involved in membrane protein vesicle trafficking, and then summarizes the different plant vesicle trafficking pathways for membrane proteins. Based on the review, we systematically consolidated the chemical treatments and mutants used in the study of plant vesicle trafficking. Finally, future directions for research in vesicle trafficking pathways of plant membrane proteins are discussed. With recent advancements, we expect major breakthroughs in our understanding of the regulatory mechanisms that determine the perception and adaptability of plants to their environments in the coming years.

Ursolic acid-piperazine-dithiocarbamate ruthenium(II) polypyridyl complexes induced necroptosis in MGC-803 cells.

Three ursolic acid-piperazine-dithiocarbamate ruthenium(II) polypyridyl complexes Ru1-Ru3 were designed and synthesized for evaluating antitumor activity. All the complexes exhibited high in vitro cytotoxicity against MGC-803, T24, HepG2, CNE2, MDA-MB-231, MCF-7, A549, and A549/DDP cell lines. Ru1, Ru2, and Ru3 were 11, 8 and 10 times, respectively, more active than cisplatin against A549/DDP. An in vivo study on MGC-803 xenograft mouse models demonstrated that representative Ru2 exhibited an effective inhibitory effect on tumor growth, showing stronger antitumor activity than cisplatin. Biological investigations suggested that Ru2 entered MGC-803 cells by a clathrin-mediated endocytic pathway, initially localizing in the lysosomes and subsequently escaping and localizing in the mitochondria. Mitochondrial swelling resulted in vacuolization, which induced vacuolation-associated cell death and necroptosis with the formation of necrosomes (RIP1-RIP3) and the uptake of propidium iodide. These results demonstrate that the potential of Ru2 as a chemotherapeutic agent to kill cancer cells via a dual mechanism represents an alternative way to eradicate apoptosis-resistant forms of cancer.

Novel pH-sensitive nanoparticles based on prodrug strategy to delivery All-Trans Retinoic Acid for breast cancer

Developing chemotherapy with nanoparticle-based prodrugs provides promising strategies for improving the safety and delivery of anti-cancer drugs therapeutics and effective cancer treatment. Herein, we developed a pH-sensitive prodrug delivery system (All-Trans-Retinoic Acid (ATRA) grafted poly (β-amino esters) (PBAE) copolymers, ATRA-g-PBAE) for delivery of ATRA with some physicochemical and biological properties. The in vitro release of ATRA-g-PBAE prodrug nanoparticles (PNPs) was sustained-release and pH-sensitive. The cytotoxicity and uptake of different preparations in vitro were evaluated on MCF-7 cells at pH 7.4 and 5.5. The carrier PBAE had no cytotoxicity, and ATRA-g-PBAE PNPs could significantly inhibit cell growth at pH 5.5. MCF-7 cells treated with Cy5.5 grafted PBAE (Cy5.5-PBAE) showed stronger fluorescence signals at pH 5.5. Meanwhile, ATRA-g-PBAE PNPs entered the cell via a clathrin-mediated endocytic pathway. Subsequently, PBAE protonation facilitated the escape of PNPs from the lysosome and released the drug. ATRA-g-PBAE seems promising as a novel pH-sensitive prodrug to overcome the limitations of ATRA for breast cancer therapy.

Vibropolyfection: coupling polymer-mediated gene delivery to mechanical stimulation to enhance transfection of adherent cells

BackgroundWith the success of recent non-viral gene delivery-based COVID-19 vaccines, nanovectors have gained some public acceptance and come to the forefront of advanced therapies. Unfortunately, the relatively low ability of the vectors to overcome cellular barriers adversely affects their effectiveness. Scientists have thus been striving to develop ever more effective gene delivery vectors, but the results are still far from satisfactory. Therefore, developing novel strategies is probably the only way forward to bring about genuine change. Herein, we devise a brand-new gene delivery strategy to boost dramatically the transfection efficiency of two gold standard nucleic acid (NA)/polymer nanoparticles (polyplexes) in vitro.ResultsWe conceived a device to generate milli-to-nanoscale vibrational cues as a function of the frequency set, and deliver vertical uniaxial displacements to adherent cells in culture. A short-lived high-frequency vibrational load (t = 5 min, f = 1,000 Hz) caused abrupt and extensive plasmalemma outgrowths but was safe for cells as neither cell proliferation rate nor viability was affected. Cells took about 1 hr to revert to quasi-naïve morphology through plasma membrane remodeling. In turn, this eventually triggered the mechano-activated clathrin-mediated endocytic pathway and made cells more apt to internalize polyplexes, resulting in transfection efficiencies increased from 10-to-100-fold. Noteworthy, these results were obtained transfecting three cell lines and hard-to-transfect primary cells.ConclusionsIn this work, we focus on a new technology to enhance the intracellular delivery of NAs and improve the transfection efficiency of non-viral vectors through priming adherent cells with a short vibrational stimulation. This study paves the way for capitalizing on physical cell stimulation(s) to significantly raise the effectiveness of gene delivery vectors in vitro and ex vivo.Graphical

Use of Fluorescent 2-AB to Explore the Bidirectional Transport Mechanism of Pseudostellaria heterophylla Polysaccharides across Caco-2 Cells.

Polysaccharides are abundant in natural resources and perform numerous physiological functions. Polysaccharide structures often lack chromophore groups; thus, current analytical methods cannot distinguish polysaccharide metabolites in the body or polysaccharide prototypes in biological samples. Thus, the measurement of polysaccharides in blood, bodily fluid, and cell-culture medium is difficult. Our early-stage research resulted in the isolation of two homogeneous polysaccharides from Pseudostellaria heterophylla, PHP0.5MSC-F and PHPH-1-2, which have anti-hyperglycemia and insulin resistance improvement effects for type 2 diabetes. In this study, the reducing terminal sugars of PHP0.5MSC-F and PHPH-1-2 were labeled with 2-aminobenzamide (2-AB) to prepare novel fluorescent probes for HPLC-coupled fluorescence detection (HPLC-FLD). Quantitative analysis was performed in reference to T40, and the detection limit for PHP0.5MSC-F was found to be 8.84 μg/mL with a linear range of 29.45–683.28 μg/mL. In reference to T70, the detection limit for PHPH-1-2 was found to be 13.89 μg/mL with a linear range of 46.29–462.76 μg/mL. This method was used to measure the bidirectional transport of polysaccharides across caco-2 cells from apical to basolateral (AP→BL) or from basolateral to apical (BL→AP) directions and to evaluate the polysaccharide bioavailability. The drug absorption capacity was determined based on the apparent permeability coefficient (Papp), and the Papp values for the two polysaccharides were found to be greater than 1 × 10−6 cm/s, which suggests easy absorption. Regarding bidirectional transport, the AP→BL Papp values were greater than the BL→AP values; thus, PHP0.5MSC-F and PHPH-1-2 mainly underwent passive transference. The two membrane permeable polysaccharides were not P-gp efflux transporter substrates. The absorption mechanism of PHP0.5MSC-F complies with passive diffusion under a concentration gradient, whereas PHPH-1-2 mainly utilizes a clathrin-mediated endocytic pathway to enter caco-2 cells. This innovative HPLC-FLD method can help to track polysaccharide internalization in vitro and in vivo to facilitate cellular uptake and biodistribution exploration.

FGF Exhibits an Important Biological Role on Regulating Cell Proliferation of Breast Cancer When it Transports Into The Cell Nuclei.

The endocrine system is closely related to the development of the breast cancer. Many studies have shown that FGF1 (Fibroblast growth factor-1) is involved the occurrence and development of the breast cancer. But up to now, the cellular behavior and characteristics of FGF1 in breast cancer have not been fully revealed. In the current study, breast cancer cell was used as an in vitro cell model to investigate FGF's cell property. The results showed that FGF1 internalized into cells in a time-dependent manner. Further study indicated that both clathrin-mediated and caveolin-mediated endocytic pathway are involved in the internalization of FGF/FGFR (Fibroblast growth factor receptor), and both clathrin-mediated endocytosis and caveolin-mediated endocytosis are involved in the process of FGF1's nuclear localization. Further study showed that Rab5 also plays an important role in the process of nuclear localization of FGF-1. In addition, we found that FGF1 and FGFR transported to the cell nuclei of breast cancer. Further experimental results indicated that the nuclear-localized FGF1 and/or FGFR is closely associated to cell proliferation of breast cancer cell. Taken together, the current work lays the foundation for exploring the relationship between nuclear-localized FGF1/FGFR and the occurrence and development of breast cancer.

Iron Uptake in Drosophila Sg4 Cells Does Not Require Endocytosis

Iron is essential for all organisms including insects; however, at high levels it is toxic. Therefore, the transport and uptake of iron must be well-regulated, and in insects this process is poorly understood. In mammals, the most widely known mechanism of iron uptake is receptor-mediated endocytic uptake of Fe3+-transferrin. In the acidified endosome, iron is released from transferrin, reduced by a ferric reductase to Fe2+, and transported across the endosome membrane through divalent metal transporter 1 (DMT1). The goal of this project was to determine whether iron uptake by cultured Drosophila Sg4 cells (an isolate of embryonically derived S2 cells) requires endocytosis. To test this hypothesis, we blocked endocytosis and then measured cellular iron content. We performed RNAi-mediated knockdown of rab5, which is expected to block both clathrin-mediated and caveolar endocytic pathways, and we also treated cells with one of three chemical inhibitors: 200 nM bafilomycin-A1, which inhibits clathrin-mediated endocytosis in S2 cells, 5 mM methyl-beta-cyclodextrin, which blocks caveolae-mediated endocytosis, and 1 µM cytochalasin D, which blocks phagocytosis and macropinocytosis. The rab5 RNAi and chemical treatments caused a slight decrease in cell growth and viability, but they did not decrease iron content. The results of these experiments suggest that endocytosis is not required for iron uptake by Sg4 cells. To test our hypothesis using a different strategy, we are evaluating the role in iron uptake of an insect homolog of DMT1, Malvolio (Mvl). Based on previous studies, Mvl appears to be a divalent metal transporter that is present mainly in endosome membranes. We used RT-PCR to verify that Sg4 cells express Mvl, and we are currently performing RNAi to assess the effect of Mvl knockdown on cellular iron content. Given that blocking endocytosis had no effect on cellular iron content, if Mvl participates in endocytic uptake of protein-bound iron, we expect that knockdown of Mvl will also have no effect on cellular iron content.

Cyclodextrin-Based Nanostructure Efficiently Delivers siRNA to Glioblastoma Cells Preferentially via Macropinocytosis.

Small interfering ribonucleic acid (siRNA) has the potential to revolutionize therapeutics since it can knockdown very efficiently the target protein. It is starting to be widely used to interfere with cell infection by HIV. However, naked siRNAs are unable to get into the cell, requiring the use of carriers to protect them from degradation and transporting them across the cell membrane. There is no information about which is the most efficient endocytosis route for high siRNA transfection efficiency. One of the most promising carriers to efficiently deliver siRNA are cyclodextrin derivatives. We have used nanocomplexes composed of siRNA and a β-cyclodextrin derivative, AMC6, with a very high transfection efficiency to selectively knockdown clathrin heavy chain, caveolin 1, and p21 Activated Kinase 1 to specifically block clathrin-mediated, caveolin-mediated and macropinocytosis endocytic pathways. The main objective was to identify whether there is a preferential endocytic pathway associated with high siRNA transfection efficiency. We have found that macropinocytosis is the preferential entry pathway for the nanoparticle and its associated siRNA cargo. However, blockade of macropinocytosis does not affect AMC6-mediated transfection efficiency, suggesting that macropinocytosis blockade can be functionally compensated by an increase in clathrin- and caveolin-mediated endocytosis.

GRK2-mediated receptor phosphorylation and Mdm2-mediated β-arrestin2 ubiquitination drive clathrin-mediated endocytosis of G protein-coupled receptors

Clathrin-mediated and caveolar endocytic pathways represent the major routes through which G protein-coupled receptors (GPCRs) could be internalized. GPCR kinase 2 (GRK2) and β-arrestins are representative proteins that mediate the GPCR endocytosis. However, the molecular mechanisms through which GRK2 and β-arrestin mediate clathrin-mediated and caveolar endocytosis remain unclear. In this study, we determined the cellular components and processes that mediate the selective interaction between clathrin/caveolin1 and GRK2/β-arrestins. For this we utilized the following: (i) mutant dopamine D2 receptor and β2 adrenoceptor in which the potential GRK2 phosphorylation sites were altered and (ii) cells in which clathrin, caveolin1, β-arrestins, or Mdm2 expression were knocked down. Our results showed that clathrin-mediated endocytosis occurs more rapidly than caveolar endocytosis. Clathrin-mediated endocytosis and the interaction between clathrin and GRK2/β-arrestin2 occurred in a GRK2-mediated receptor phosphorylation-dependent manner. In contrast, caveolar endocytosis and the interaction between caveolin1 and GRK2/β-arrestin2 were independent of receptor phosphorylation status. Mdm2-mediated ubiquitination of β-arrestin, which occurred in a receptor phosphorylation-dependent manner, was required for the interaction of arrestin with clathrin. Thus, this study shows that GRK2-mediated receptor phosphorylation accompanied by β-arrestin ubiquitination is a critical cellular event that links GRK2 and β-arrestins to clathrin-mediated endocytosis.

Mechanism Investigation of Hyaluronidase-Combined Multistage Nanoparticles for Solid Tumor Penetration and Antitumor Effect.

BackgroundHyaluronic acid (HA) is a major component of extracellular matrix (ECM) and its over expression in tumor tissues contributes to the increase of interstitial fluid pressure (IFP) and hinders the penetration of nanoparticles into solid tumors.Materials and MethodsWe here reported a tumoral microenvironment responsive multistage drug delivery system (NPs-EPI/HAase) which was formed layer by layer via electrostatic interaction with epirubicin (EPI)-loaded PEG-b-poly(2-(diisopropylamino)ethyl methacrylate)-b-poly(2-guanidinoethylmethacrylate) (mPEG-PDPA-PG, PEDG) micelles (NPs-EPI) and hyaluronidase (HAase). In this paper, we focused on the hyaluronidase-combined nanoparticles (NPs-EPI/HAase) for tumor penetration in tumor spheroid and solid tumor models in vitro and in vivo.ResultsOur results showed that NPs-EPI/HAase effectively degrade the HA in ECM and facilitate deep penetration of NPs-EPI into solid tumor. Moreover, NPs-EPI mainly employed clathrin-mediated and macropinocytosis-mediated endocytic pathways for cellular uptake and were subsequently directed to the lysosomes for further drug release triggered by proton sponge effect. Compared with NPs-EPI, the HAase coating group showed an enhanced tumoral drug delivery efficacy and inhibition of tumor growth.ConclusionOverall, our studies demonstrated that coating nanoparticles with HAase can provide a simple but efficient strategy for nano-drug carriers to enhance solid tumor penetration and chemotherapeutic efficacy.

Fluorescent Polymer Dot-Based Multicolor Stimulated Emission Depletion Nanoscopy with a Single Laser Beam Pair for Cellular Tracking.

Stimulated emission depletion (STED) nanoscopy provides subdiffraction resolution while preserving the benefits of fluorescence confocal microscopy in live-cell imaging. However, there are several challenges for multicolor STED nanoscopy, including sophisticated microscopy architectures, fast photobleaching, and cross talk of fluorescent probes. Here, we introduce two types of nanoscale fluorescent semiconducting polymer dots (Pdots) with different emission wavelengths: CNPPV (poly[2-methoxy-5-(2-ethylhexyloxy)-1,4-(1-cyanovinylene-1,4-phenylene)]) Pdots and PDFDP (poly[{9,9-dihexyl-2,7-bis(1-cyanovinylene)fluorene}-alt-co-{2,5-bis (N,N'-diphenylamino)-1,4-phenylene}]) Pdots, for dual-color STED bioimaging and cellular tracking. Besides bright fluorescence, strong photostability, and easy bioconjugation, these Pdots have large Stokes shifts, which make it possible to share both excitation and depletion beams, thus requiring only a single pair of laser beams for the dual-color STED imaging. Long-term tracking of cellular organelles by the Pdots has been achieved in living cells, and the dynamic interaction of endosomes derived from clathrin-mediated and caveolae-mediated endocytic pathways has been monitored for the first time to propose their interaction models. These results demonstrate the promise of Pdots as excellent probes for live-cell multicolor STED nanoscopy.

Novel nanococktail of a dual PI3K/mTOR inhibitor and cabazitaxel for castration-resistant prostate cancer.

Prognosis of castration-resistant prostate cancer (CRPC) carries is poor, and no effective therapeutic regimen is yet known. The phosphatidylinositol-3-kinase (PI3K)/protein kinase B (Akt)/mammalian target of rapamycin (mTOR) pathway played a predominant role and may be a promising molecular target for CRPC. However, the toxicity of the dual PI3K inhibitors in clinical trials limits their clinical efficacy for CRPC. To solve this problem, we employed a highly integrated precision nanomedicine strategy to molecularly and physically target CRPC through synergistic effects, enhanced targeted drug delivery efficiency, and reduced unwanted side-effects. Gedatolisib (Ge), a potent inhibitor of PI3K/mTOR, was formulated into our disulfied-crosslinked micelle plateform (NanoGe), which exhibits excellent water solubility, small size (23.25±2 nm), excellent stability with redox stimulus-responsive disintegration, and preferential uptake at tumor sites. NanoGe improved the anti-neoplastic effect of free Ge by 53 times in PC-3M cells and 13 times in C4-2B cells though its enhanced uptake via caveolae- and clathrin-mediated endocytic pathways and the subsequent inhibition of the PI3K/mTOR pathway, resulting in Bax/Bcl-2 dependent apoptosis. In an animal xenograft model, NanoGe showed superior efficacy than free Ge, and synergized with nanoformulated cabazitaxel (NanoCa) as a nanococktail format to achieve a cure rate of 83%. Taken together, our results demonstrate the potency of NanoGe in combination with NanoCa is potent against prostate cancer.

KDEL receptor is a cell surface receptor that cycles between the plasma membrane and the Golgi via clathrin-mediated transport carriers.

KDEL receptor cycles between the ER and the Golgi to retrieve ER-resident chaperones that get leaked to the secretory pathway during protein export from the ER. Recent studies have shown that a fraction of KDEL receptor may reside in the plasma membrane and function as a putative cell surface receptor. However, the trafficking itinerary and mechanism of cell surface expressed KDEL receptor remains largely unknown. In this study, we used N-terminally Halo-tagged KDEL receptor to investigate its endocytosis from the plasma membrane and trafficking itinerary of the endocytosed receptor through the endolysosomal compartments. Our results indicate that surface-expressed KDEL receptor undergoes highly complex recycling pathways via the Golgi and peri-nuclear recycling endosomes that are positive for Rab11 and Rab14, respectively. Unexpectedly, KDEL receptor appears to preferentially utilize clathrin-mediated endocytic pathway as well as clathrin-dependent transport carriers for export from the trans-Golgi network. Taken together, we suggest that KDEL receptor may be a bona fide cell surface receptor with a complex, yet well-defined trafficking itinerary through the endolysosomal compartments.

Evaluation of Anticancer and Anti-Mitotic Properties of Quinazoline and Quinazolino-Benzothiadiazine Derivatives.

Cancer is one of the major health and social-economic problems despite considerable progress in its early diagnosis and treatment. Owing to the emergence and increase of multidrug resistance to various conventional drugs, and the continuing importance of health-care expenditure, many researchers have focused on developing novel and effective anticancer compounds. Chemical repositories provide a good platform to evaluate and exploit known chemical entities for the identification of other biological activities. In the present study, we have selected an in-house library of synthesized compounds based on two different pharmacophoric scaffolds to evaluate their cytotoxic potency on various cancer cell lines and mechanisms of action. A series of in-house synthesized quinazoline and quinazolino-benzothiadiazine derivatives were investigated for their anticancer efficacy against a panel of five cancer (DU145, MCF7, HepG2, SKOV3 and MDA-MB-231) and one normal (MRC5) cell lines. Furthermore, the active compound of the study was investigated to elucidate the mechanism of cytotoxicity by performing series of experiments such as cell cycle analysis, inhibition of tubulin polymerization, alteration of mitochondrial membrane potential, determination of endocytic pathway for drug uptake pathway and combination drug treatment. Among all the tested compounds, fifteen of them exhibited promising growth-inhibitory effect (0.15- 5.0μM) and induced cell cycle arrest in the G2/M phase. In addition, the selected compounds inhibited the microtubule assembly; altered mitochondrial membrane potential and enhanced the levels of caspase-9 in MCF-7 cells. Furthermore, the active compound with a combination of drugs showed a synergistic effect at lower concentrations, and the drug uptake was mediated through clathrin-mediated endocytic pathway. Our results indicated that quinazoline and quinazolino-benzothiadiazine conjugates could serve as potential leads in the development of new anticancer agents.

Molecular characterization of clathrin heavy chain (Chc) in Rhipicephalus haemaphysaloides and its effect on vitellogenin (Vg) expression via the clathrin-mediated endocytic pathway.

Clathrin plays an important role in arthropods, but its function in ticks has not been explored. Here, we describe the molecular characteristics of the clathrin heavy chain of the tick Rhipicephalus haemaphysaloides and its effects on yolk development. The open reading frame of the clathrin heavy chain (Chc) (Rh-Chc) gene consists of 5103 nucleotides encoding 670 amino acids, which is most closely related to that of Ixodes scapularis and relatively close to Homo sapiens and Drosophila melanogaster. Real-time qPCR revealed that Rh-Chc was expressed at all developmental stages and organs. After Rh-Chc is silenced, ticks did not feed and mortality rate was 100%. Moreover, Rh-Chc co-localized with Vitellogenin receptor (VgR) on oocyte membrane. Immunofluorescence showed that the expression of Vitellogenin (Vg) (Rh-Vg) was also closely related to Rh-Chc. Immunofluorescence showed that the expression of Vg was also closely related to Rh-Chc, Rh-Chc silencing slowed the development of oocytes in tick, and culture of ovary in vitro silenced Rh-Chc, the development of oocytes in ticks also slowed down. Overall, the results of this study indicated that Rh-Chc is a vital gene in the tick R. haemaphysaloides that plays an important role in its growth, development, and reproduction.

Mechanism of internalization of MDA-7/IL-24 protein and its cognate receptors following ligand-receptor docking.

Melanoma differentiation associated gene-7 (mda-7/IL-24) is a member of the IL-10 family of cytokines, with ubiquitous direct and “bystander” tumor-selective killing properties. MDA-7/IL-24 protein binds distinct type II cytokine heterodimeric receptor complexes, IL-20R1/IL-20R2, IL-22R1/IL-20R1 and IL-22R1/IL-20R2. Recombinant MDA-7/IL-24 protein induces endogenous mda-7/IL-24 expression in a receptor-dependent manner; since A549 cells that lack a complete set of cognate receptors are not responsive to exogenous protein. The mechanism of MDA-7/IL-24 ligand-receptor biology is not well understood. We explored the interaction of MDA-7/IL-24 with its’ receptors and the consequences of ligand-receptor docking. Using both pharmacological and genetic approaches we demonstrate that MDA-7/IL-24 internalization employs the clathrin-mediated endocytic pathway leading to degradation of receptors via the lysosomal/ubiquitin proteosomal pathway. This clathrin-mediated endocytosis is dynamin-dependent. This study resolves a novel mechanism of MDA-7/IL-24 protein “bystander” function, which involves receptor/protein-mediated internalization and receptor degradation.

Synthesis and engineering of mesoporous ZnO@HAP heterostructure as a pH-sensitive nano-photosensitizer for chemo-photodynamic therapy of malignant tumor cells

The aim of this study was the synthesis of a platform with an optimum physicochemical property and photocatalytic activity for delivery of a standard chemotherapy drug, doxorubicin (Dox). The mesoporous rod-shaped zinc oxide/hydroxyapatite (ZnO@HAP) heterostructures were successfully fabricated and characterized by XRD, FT-IR, FE-SEM, and EDX, and then the nanocomposites with well-defined properties were selected as a nanocarrier for the delivery of Dox. The drug delivery system was decorated with F127 and folic acid (FA), providing a water-soluble system in aqueous medium and targeting effects against tumor cells, respectively. Afterward, Dox was loaded on the surface of the nanocomposites through a single-emulsion solvent-evaporation method. Drug release assessment revealed that proteases cause the release of Dox under a low pH condition, imitating the intracellular compartments like the lysosomes. Based on the biological studies, Dox&FA.F127.ZnO@HAP exerted the most efficient cytotoxic activity against the tumor cells as it was along with the UVA treating. Moreover, it was found that Dox&FA.F127.ZnO@HAP possesses higher cellular uptake efficiency relative to free Dox, particularly in folate-overexpressed tumor cells through clathrin-mediated endocytic pathway. In addition, co-treatment of the tumor cells with Dox&FA.F127.ZnO@HAP and UVA intensified the production of intracellular ROS and drastically enhanced cellular death with mode of apoptosis.

Entry of Challenge Virus Standard (CVS) -11 into N2a cells via a clathrin-mediated, cholesterol-, dynamin-, pH-dependent endocytic pathway

BackgroundRabies virus (RABV), a member of Lyssavirus of Rhabdoviridae family, is a kind of negative-strand RNA virus. The zoonosis caused by RABV leads to high mortality in animals and humans. Though with the extensive investigation, the mechanisms of RABV entry into cells have not been well characterized.MethodsChemical inhibitors and RNA interference (RNAi) were used to analysis RABV internalization pathway. The expression level of viral N protein was examined by quantitative PCR and western blot, and the virus infection in the cells was visualized by fluorescence microscopy.ResultsWe firstly examined the endocytosis pathway of the challenge virus standard (CVS) -11 strain in N2a cells. Chlorpromazine treatment and knockdown of clathrin heavy chain (CHC) significantly reduced viral entry, which proved clathrin was required. Meanwhile neither nystatin nor knocking down caveolin-1 (Cav1) in N2a cells had an effect on CVS-11 infection, suggesting that caveolae was independent for CVS-11 internalization. And when cholesterol of cell membrane was extracted by MβCD, viral infection was strongly impacted. Additionally by using the specific inhibitor dynasore and ammonium chloride, we verified that dynamin and a low-pH environment were crucial for RABV infection, which was confirmed by confocal microscopy.ConclusionsOur results demonstrated that CVS-11 entered N2a cells through a clathrin-mediated, cholesterol-, pH-, dynamin-required, and caveolae-independent endocytic pathway.