Macropinocytosis Inhibitor Research Articles

Quantitative Assessment of Macropinocytosis in mTORC1-Hyperactive Cells using Flow Cytometry.

Macropinocytosis is a highly conserved, actin-dependent endocytic process that allows the uptake of extracellular material, including proteins and lipids. In proliferating cells, macropinocytosis can deliver extracellular nutrients to the lysosome, processed into critical macromolecule building blocks. Recent studies have highlighted the dependence of multiple cancers on macropinocytosis, including breast, colorectal and pancreatic cancer. Ras mutations are thought to be the driver events behind macropinocytosis initiation, leading to the activation of cellular anabolic processes via the mTORC1 signaling pathway. Interestingly, mTORC1 can also be activated by macropinocytosis independently of Ras. Therefore, macropinocytosis represents a metabolic vulnerability that can be leveraged to target macropinocytic tumors by limiting their access to nutrients therapeutically. In Tuberous Sclerosis Complex (TSC) and Lymphangioleiomyomatosis (LAM), mTORC1-hyperactivation leads to enhanced macropinocytosis and metabolic reprogramming. Here, we describe a flow cytometry-based protocol to assess macropinocytosis in mammalian cells quantitatively. TSC2-deficient MEFs are employed, which exhibit aberrant activation of mTORC1 and have been shown to have increased macropinocytosis compared to TSC2-expressing cells. Cells treated with pharmacologic inhibitors of macropinocytosis are incubated with fluorescently labeled, lysine-fixable, 70 kDa dextran, or fluorescently labeled bovine serum albumin (BSA) assayed by flow cytometry. To date, robust image-based techniques have been developed to quantitatively assess macropinocytosis in tumor cells in vitro and in vivo. This analysis provides a quantitative assessment of macropinocytosis in multiple experimental conditions and complements existing image-based techniques.

Enhancing Proteasome-Inhibitory Activity and Specificity of Bortezomib By Endocytosis-Mediated Uptake of CD38 Targeted Nanoparticles in Multiple Myeloma

The establishment of more effective treatments that can circumvent chemoresistance in Multiple Myeloma (MM) is a priority. Although bortezomib (BTZ) is one of the most potent proteasome inhibitors available, still possesses limitations related to dose limiting side effects. Several strategies have been developed to improve the delivery of chemotherapies to MM by targeting different moieties expressed on MM cells to nanoparticle delivery systems (NPs), which have failed mainly due to their heterogeneous expression on these cells. Our goal was to test CD38 targeted chitosan NPs as novel targeting moiety for MM to improve the potency and efficacy of BTZ in MM cells and reduce the side effects in healthy tissue. Due to its high expression on MM cells, CD38 is used as a marker for identification of MM cells, and there are several indications supporting the notion that CD38 plays significant roles in the progression of MM.We have fine-tuned the production of anti-CD38 chitosan NPs with a small size (50nm) to allow better penetration to tumors, and we showed that these NPs were stable at different storage conditions and periods. The chitosan NPs showed preferential drug release in MM tumor microenvironment compared to normal tissue microenvironment, and this release was pH dependent.The uptake of the anti-CD38 chitosan NPs to MM cells (cell lines and primary) was significantly higher than their uptake in normal cells (4-fold of BM MNCs, and 10-fold of PB MNCs) demonstrating that the anti-CD38 chitosan NPs are selective and specific to MM. BTZ-loaded anti-CD38 chitosan NPs demonstrated enhanced cytotoxicity and apoptosis on MM cells compared to similar concentration of BTZ as free drug; demonstrated by induction of more profound cell cycle arrest and apoptosis leading to cell death through inactivation of proliferative cell signaling pathways (MAPK, PI3K) and activation of pro-apoptotic pathways (PARP and Caspase-3). We also found that BTZ-loaded anti-CD38 NPs proved to be more efficacious than free BTZ and overcame CAM-DR and hypoxia-mediated drug resistance. The use of a 3DTEBM model, which recreate MM pathophysiology, corroborated the increase cytotoxicity of anti-CD38 chitosan NPs on MM cells relative to free drug and allowed to corroborated co-localization of the NPs in the MM cells.We found that BTZ-loaded NPs were significantly more potent in the proteasome activity inhibition than free BTZ and correlated with the boron content in MM cells treated with BTZ-loaded anti-CD38 NPs, which was 3-fold higher than in MM cells treated with BTZ as free drug. To confirm that the process involved in the internalization of the anti-CD38 chitosan NPs was endocytosis, we have shown that the NPs distribution in the cells co-localized with the distribution of endosomes and found that the anti-CD38 NPs were uptaken through clathrin-mediated and caveolae-mediated early endocytosis which progressed to late endocytosis and further delivery of the NPs content into the cells. Finally, we linked the internalization through endocytic pathways of anti-CD38 NPs to the enhanced proteasome activity inhibition of these NPs in MM cells. Treatment of MM cells with clathrin and caveolae-mediated early endocytosis, or late endocytosis inhibitors (but not macropinocytosis inhibitors) significantly reduced the proteasome inhibition of BTZ-loaded anti-CD38 chitosan NPs. When BTZ is administered as a free drug, it penetrated through passive diffusion into MM cells as well as in normal cells, and inhibition of the different endocytosis routes did not induce any change in its ability to inhibit the proteasome.In vivo studies showed that treatment with BTZ-loaded anti-CD38 targeted NPs (1mg/kg once a week) showed a markedly improved tumor growth inhibition, improved overall survival, and reduction of side effects compared to non-targeted NPs and free drug.In conclusion, anti-CD38 chitosan NPs showed preferential BTZ release in tumor-microenvironment, specific binding to MM cells, and an improved drug cellular uptake through endocytosis, which translated in enhanced proteasome inhibition and robust cytotoxic effect on MM cells. Furthermore, the anti-CD38 chitosan NPs specifically delivered therapeutic agents to MM cells improving therapeutic efficacy and reducing side effects in vivo. We are the first to report the enhancement of the efficacy and specificity of proteasome inhibitors due to endocytosis-driven delivery of NPs. DisclosuresDe La Puente:Cellatrix LLC: Other: Co-Founder. Vij:Celgene, Onyx, Takeda, Novartis, BMS, Sanofi, Janssen, Merck: Consultancy; Takeda, Onyx: Research Funding. Azab:Glycomimetics: Research Funding; Selexys: Research Funding; Karyopharm: Research Funding; Cellatrix LLC: Other: Founder and owner; Verastem: Research Funding; Cell Works: Research Funding; Abbvie: Research Funding; Tioma: Research Funding; Cleave Bioscience: Research Funding; Targeted Therapeutics LLC: Other: Founder and owner.

KRAS-Mutated Myeloma Cells Exploit Autophagy and Macropinocytosis to Tolerate Glutamine-Deprived Conditions

Introduction: Sustained cancer cell growth requires up-regulation of nutrient acquisition mechanisms. Novel approaches can identify “tumor fuel”, the way it is obtained and then altered for cellular usage. Autophagy is a conserved catabolic process that is thought to replenish nutrient pool and thereby starvation survival by providing bioenergetic and biosynthetic substrates. Some KRAS-mutated cancers show heightened dependency to glutamine as a source of energy. Since a large proportion of myeloma tumors are KRAS-mutated, we investigated in the present study the contribution of macropinocytosis and autophagy as the cellular mechanisms involved in protein uptake and degradation, respectively, to growth and survival of myeloma cells under glutamine-deprived condition.Methods: Macropinosomes were visualized using confocal microscopy in KRAS-mutated and WT human myeloma cells utilising TMR-dextran and 5-[N-ethyl-N-isopropyl] amiloride (EIPA) as a marker and specific inhibitor of macropinocytosis, respectively. Internalization and degradation of labelled albumin and its co-localization with TMR-dextran were evaluated in parallel. Bafilomycin was employed to evaluate the dependency of protein degradation on autophagy. Cell sensitivity to glutamine deprivation was examined with an ATP-based viability test. To evaluate the possible role of macropinocytosis and autophagy at glutamine deprivation conditions, the effect of albumin supplementation on cell growth and viability was studied in the presence or absence of EIPA or chloroquine. To test whether the degradation of macropinocytosed albumin results in the production of intracellular glutamine, the intracellular concentrations of glutamine was directly measured.Results: KRAS-mutated KMS28-PE, KMS18 and MM1S cells showed higher levels of EIPA-sensitive macropinocytosis compared to WT-KRAS KMS34, KMS12-BM and TK1 cells. Macropinocytosis led to the internalization of albumin which subsequently underwent bafilomycin-sensitive proteolytic degradation in KRAS-mutated cells. The viability of WT-KRAS cells was reduced in sub-physiological concentrations of glutamine when compared with KRAS-mutated cells that not only remained viable but could continue to slowly proliferate over a period of 7 days (p˂0.05). The compromised growth rate of KRAS-mutated cells at very low concentrations of glutamine was rescued by albumin and this effect was abrogated by EIPA or chloroquine (p˂0.05). The KRAS-mutated cells demonstrated an EIPA-sensitive increase in intracellular concentration of glutamine following albumin supplementation (p˂0.05) that was abrogated following the treatment with chloroquine. In WT cells, in contrast, the level of intracellular glutamine remained almost unchanged following albumin supplementation.Conclusions: Autophagy and macropinocytosis maintain cellular growth and survival in the context of nutrient deprivation by provision of glutamine, and potentially other amino acids, in RAS-mutated myeloma cancer. These data suggest that novel anti-cancer strategies interrupting these cellular mechanisms may represent a promising therapeutic approach to myeloma. DisclosuresSpencer:Janssen: Honoraria, Research Funding; Amgen: Consultancy, Honoraria, Research Funding.

Visualizing Membrane Ruffle Formation using Scanning Electron Microscopy.

Membrane ruffling is the formation of motile plasma membrane protrusions containing a meshwork of newly polymerized actin filaments. Membrane ruffles may form spontaneously or in response to growth factors, inflammatory cytokines, and phorbol esters. Some of the membrane protrusions may reorganize into circular membrane ruffles that fuse at their distal margins and form cups that close and separate into the cytoplasm as large, heterogeneous vacuoles called macropinosomes. During the process, ruffles trap extracellular fluid and solutes that internalize within macropinosomes. High-resolution scanning electron microscopy (SEM) is a commonly used imaging technique to visualize and quantify membrane ruffle formation, circular protrusions, and closed macropinocytic cups on the cell surface. The following protocol describes the cell culture conditions, stimulation of the membrane ruffle formation in vitro, and how to fix, dehydrate, and prepare cells for imaging using SEM. Quantification of membrane ruffling, data normalization, and stimulators and inhibitors of membrane ruffle formation are also described. This method can help answer key questions about the role of macropinocytosis in physiological and pathological processes, investigate new targets that regulate membrane ruffle formation, and identify yet uncharacterized physiological stimulators as well as novel pharmacological inhibitors of macropinocytosis.

Cancer cells escape autophagy inhibition via NRF2-induced macropinocytosis.

Many cancers, including pancreatic ductal adenocarcinoma (PDAC), depend on autophagy-mediated scavenging and recycling of intracellular macromolecules, suggesting that autophagy blockade should cause tumor starvation and regression. However, until now autophagy-inhibiting monotherapies have not demonstrated potent anti-cancer activity. We now show that autophagy blockade prompts established PDAC to upregulate and utilize an alternative nutrient procurement pathway: macropinocytosis (MP) that allows tumor cells to extract nutrients from extracellular sources and use them for energy generation. The autophagy to MP switch, which may be evolutionarily conserved and not cancer cell restricted, depends on activation of transcription factor NRF2 by the autophagy adaptor p62/SQSTM1. NRF2 activation by oncogenic mutations, hypoxia, and oxidative stress also results in MP upregulation. Inhibition of MP in autophagy-compromised PDAC elicits dramatic metabolic decline and regression of transplanted and autochthonous tumors, suggesting the therapeutic promise of combining autophagy and MP inhibitors in the clinic.

SLIT2/ROBO1-signaling inhibits macropinocytosis by opposing cortical cytoskeletal remodeling

Macropinocytosis is essential for myeloid cells to survey their environment and for growth of RAS-transformed cancer cells. Several growth factors and inflammatory stimuli are known to induce macropinocytosis, but its endogenous inhibitors have remained elusive. Stimulation of Roundabout receptors by Slit ligands inhibits directional migration of many cell types, including immune cells and cancer cells. We report that SLIT2 inhibits macropinocytosis in vitro and in vivo by inducing cytoskeletal changes in macrophages. In mice, SLIT2 attenuates the uptake of muramyl dipeptide, thereby preventing NOD2-dependent activation of NF-κB and consequent secretion of pro-inflammatory chemokine, CXCL1. Conversely, blocking the action of endogenous SLIT2 enhances CXCL1 secretion. SLIT2 also inhibits macropinocytosis in RAS-transformed cancer cells, thereby decreasing their survival in nutrient-deficient conditions which resemble tumor microenvironment. Our results identify SLIT2 as a physiological inhibitor of macropinocytosis and challenge the conventional notion that signals that enhance macropinocytosis negatively regulate cell migration, and vice versa.

Cathepsin B Protease Facilitates Chikungunya Virus Envelope Protein-Mediated Infection via Endocytosis or Macropinocytosis.

Chikungunya virus (CHIKV) is an enveloped virus that enters host cells and transits within the endosomes before starting its replication cycle, the precise mechanism of which is yet to be elucidated. Endocytosis and endosome acidification inhibitors inhibit infection by CHIKV, murine leukemia virus (MLV), or SARS-coronavirus, indicating that these viral entries into host cells occur through endosomes and require endosome acidification. Although endosomal cathepsin B protease is necessary for MLV, Ebola virus, and SARS-CoV infections, its role in CHIKV infection is unknown. Our results revealed that endocytosis inhibitors attenuated CHIKV-pseudotyped MLV vector infection in 293T cells but not in TE671 cells. In contrast, macropinocytosis inhibitors attenuated CHIKV-pseudotyped MLV vector infection in TE671 cells but not in 293T cells, suggesting that CHIKV host cell entry occurs via endocytosis or macropinocytosis, depending on the cell lines used. Cathepsin B inhibitor and knockdown by an shRNA suppressed CHIKV-pseudotyped MLV vector infection both in 293T and TE671 cells. These results show that cathepsin B facilitates CHIKV infection regardless of the entry pathway.

GSK3 Inhibits Macropinocytosis and Lysosomal Activity through the Wnt Destruction Complex Machinery

SUMMARYCanonical Wnt signaling is emerging as a major regulator of endocytosis. Here, we report that Wnt-induced macropinocytosis is regulated through glycogen synthase kinase 3 (GSK3) and the β-catenin destruction complex. We find that mutation of Axin1, a tumor suppressor and component of the destruction complex, results in the activation of macropinocytosis. Surprisingly, inhibition of GSK3 by lithium chloride (LiCl), CHIR99021, or dominant-negative GSK3 triggers macropinocytosis. GSK3 inhibition causes a rapid increase in acidic endolysosomes that is independent of new protein synthesis. GSK3 inhibition or Axin1 mutation increases lysosomal activity, which can be followed with tracers of active cathepsin D, β-glucosidase, and ovalbumin degradation. Microinjection of LiCl into the blastula cavity of Xenopus embryos causes a striking increase in dextran macropinocytosis. The effects of GSK3 inhibition on protein degradation in endolysosomes are blocked by the macropinocytosis inhibitors EIPA or IPA-3, suggesting that increases in membrane trafficking drive lysosomal activity.

Uptake of collagen type I via macropinocytosis cause mTOR activation and anti-cancer drug resistance

Collagen type I (Col I) is one of the major extracellular matrix proteins in the cancer tissue. Previously, we have reported that Col I induces epidermal growth factor receptor tyrosine kinase inhibitor (EGFR-TKI) resistance by mTOR activation through Akt and ERK1/2 independent pathway. In this study, we aimed to elucidate the molecular mechanism of Col I induced EGFR-TKI resistance. First, we demonstrated the uptake of fluorescently labeled Col I by EGFR-mutated lung cancer cell line PC-9 cells using confocal microscopy and flow cytometry. Metabolome analysis revealed that the metabolic profiles of PC-9 cells was influenced by Col I treatment. Uptake of Col I into PC-9 cells was not inhibited by MMP inhibitor, GM6001, and endocytosis inhibitors, Pitstop2 and Dyngo4a; however, macropinocytosis inhibitor EIPA prevented its uptake. Moreover, the combination of EIPA and EGFR-TKI abrogated Col I-induced EGFR-TKI resistance in PC-9 cells. Inhibition of Rac1, which is essential for micropinocytosis, also decreased the uptake of Col I in PC-9 cells and restored their sensitivity to EGFR-TKI. Thus, EGFR mutated lung cancer cells could develop EGFR-TKI resistance by Col I uptake by macropinocytosis route.

Mediated pathways of exosomes uptake by stem cells of apical papilla

To evaluate the uptake of exosomes by stem cells from apical papilla (SCAP), thus to provide experimental basis for mechanism of the exosomes endocytosis by SCAP. (1) Exosomes of dental pulp stem cells (DPSCs) were isolated by hypercentrifugation combined with ultrafiltration method. The exosomes were identified by transmission electron microscopy, nanoparticle tracking analysis and western blot. (2) PKH-26 membrane labeling technology was used to mark the DPSCs derived exosomes. The labeled exosomes were co-cultured with SCAP at 37 °C as positive control group, and co-cultured with SCAP at 4 °C as the low-temperature treatment group, while the negative control group was set up. (3) Using clathrin-mediated endocytosis inhibitor chlorpromazine (CPZ, 10 μmol /L) as CPZ group, caveolae-mediated endocytosis Genistein (200 μmol/L) as Genistein group, and macropinocytosis inhibitor LY294002 (50 μmol/L) as LY294002 group to treat the SCAP respectively. Solvent control group (DMSO group) was set. Immunofluorescence staining was used to detect the red fluorescence SCAP and flow cytometry was used to analyze the proportion of SCAP labeled with red fluorescence. (1) The bilayer membrane and cup-shaped appearance of representative exosomes were observed. The peak of the size of DPSCs-derived exosomes was at 144 nm. The exosomes expressed exosomal marker proteins TSG101 and CD63, but not GAPDH which was the cellular internal control protein. (2) Immunofluorescence staining showed that after being co-cultured at 37 °C for 6 hours, red fluorescence could be detected in SCAP but it could not be detected after being co-cultured at 4 °C for 6 hours. After endocytosis inhibition, the red fluorescence in SCAP was reduced. Flow cytometry showed that the proportion of SCAP labeled with red fluorescence in positive group was 35.0%, in negative control group was 0.5%, and in solvent control group was 29.7%, in CPZ group, Genistein group and Genistein group were reduced to 13.7%, 16.6%, and 20.9%, respectively. SCAP could uptake the DPSCs derived exosomes, and low temperature could inhibit this process. The exosomes uptake of SCAP was mediated by the clathrin endocytosis pathway, caveolae-mediated endocytosis and macropinocytosis pathway.

Synthesis and Biological Evaluation of Genistein-IR783 Conjugate: Cancer Cell Targeted Delivery in MCF-7 for Superior Anti-Cancer Therapy.

The flavonoid-based natural product genistein is a biologically active compound possessing promising anti-oxidant and anti-cancer properties. Poor pharmacokinetics along with low potency limit however the therapeutic application of genistein in cancer therapy. In order to overcome those limitations and to expand its therapeutic window of efficacy, we sought to covalently attach genistein with a heptamethine cyanine dye—IR 783—for cancer cell targeting and enhanced delivery to tumors. Herein we report the synthesis, a selective detailed characterization and preliminary in vitro/in vivo biological evaluation of genistein-IR 783 conjugate 4. The conjugate 4 displayed improved potency against human breast cancer MCF-7 cells (10.4 ± 1.0 μM) as compared with the parent genistein (24.8 ± 0.5 μM) or IR 783 (25.7 ± 0.7 μM) and exhibited selective high uptake in MCF-7 as against the normal mammary gland MCF-10A cells in various assays. In the cell viability assay, conjugate 4 exhibited over threefold lower potency against MCF-10A cells (32.1 ± 1.1 μM) suggesting that the anti-cancer profile of parent genistein is significantly improved upon conjugation with the dye IR783. Furthermore, the genistein-IR783 conjugate 4 was shown to be especially accumulated in MCF-7 cancer cells by fluorescent intensity measurements and inverted fluorescence microscopy in fixed cells as well as in live cells with time via live cell confocal fluorescence imaging. The mechanism-based uptake inhibition of conjugate 4 was observed with OATPs inhibitor BSP and in part with amiloride, as a macropinocytosis inhibitor. For the first time we have shown amiloride inhibited uptake of cyanine dye by about ~40%. Finally, genistein-IR 783 conjugate 4 was shown to be localized in MCF-7 tumor xenografts of mice breast cancer model via in vivo near infrared fluorescence (NIRF) imaging. In conclusion, conjugation of genistein with cyanine dye IR783 indeed improved its pharmacological profile by cancer cell selective uptake and targeting and therefore warrants further investigations as a new anti-cancer therapeutics derived from natural product genistein.

Muscovy duck reovirus enters susceptible cells via a caveolae-mediated endocytosis-like pathway

Muscovy duck reovirus (MDRV) causes immunosuppression and results in high mortality among Muscovy ducklings. Cell entry is the first step of virus infection and represents a potential therapeutic target. However, very little is known about the mechanism by which MDRV penetrates the cells. The aim of this study was to explore the mechanism of MDRV cell entry and subsequent infection. DF-1 and Vero cells were pretreated with the inhibitors chlorpromazine (CPZ), cytochalasin D, methyl-beta-cyclodextrin (M-β-CD), genistein, dynasore, nocodazole, or NH4Cl, and then infected with MDRV. The copy number of the MDRV p10.8 gene and the expression of viral sigma A protein were determined by RT-PCR and western blot, respectively. Both sigma A expression and p10.8 gene copy number were decreased by treatment with M-β-CD, genistein, dynasore, nocodazole, and NH4Cl. In contrast, no effects on virus infection were detected when inhibitors of clathrin-mediated endocytosis or macropinocytosis were used. In addition, the colocalization between MDRV sigma A protein and caveolin-1 was evaluated by double-label immunofluorescence. Collectively, our data revealed that MDRV can enter susceptible cells through caveolin-dependent endocytosis involving dynamin and microtubules. Moreover, the acidic environment of the endosomes was found to be critical for efficient infection. Our findings provide new insights into the infection process of MDRV.

Evidence for Nanoparticle-Induced Lysosomal Dysfunction in Lung Adenocarcinoma (A549) Cells.

Background: Polystyrene nanoparticles (PNP) are taken up by primary rat alveolar epithelial cell monolayers (RAECM) in a time-, dose-, and size-dependent manner without involving endocytosis. Internalized PNP in RAECM activate autophagy, are delivered to lysosomes, and undergo [Ca2+]-dependent exocytosis. In this study, we explored nanoparticle (NP) interactions with A549 cells. Methods: After exposure to PNP or ambient pollution particles (PM0.2), live single A549 cells were studied using confocal laser scanning microscopy. PNP uptake and egress were investigated and activation of autophagy was confirmed by immunolabeling with LC3-II and LC3-GFP transduction/colocalization with PNP. Mitochondrial membrane potential, mitophagy, and lysosomal membrane permeability (LMP) were assessed in the presence/absence of apical nanoparticle (NP) exposure. Results: PNP uptake into A549 cells decreased in the presence of cytochalasin D, an inhibitor of macropinocytosis. PNP egress was not affected by increased cytosolic [Ca2+]. Autophagy activation was indicated by increased LC3 expression and LC3-GFP colocalization with PNP. Increased LMP was observed following PNP or PM0.2 exposure. Mitochondrial membrane potential was unchanged and mitophagy was not detected after NP exposure. Conclusions: Interactions between NP and A549 cells involve complex cellular processes leading to lysosomal dysfunction, which may provide opportunities for improved nanoparticle-based therapeutic approaches to lung cancer management.

Occludin endocytosis is involved in the disruption of the intestinal epithelial barrier in a mouse model of alcoholic steatohepatitis.

We aimed to investigate the involvement of the endocytosis of occludin, a key component of tight junction (TJ), in the ethanol-induced disassembly of TJ in a model of alcoholic steatohepatitis. Wild-type mice were fed an ethanol-containing or isocaloric liquid diet for 8 weeks and then assessed for liver injury (histopathology and measurement of serum enzymes), gut permeability (in vivo lactulose/mannitol and ex vivo dye leakage assays), intestinal epithelium ultrastructure (transmission electron microscopy), and intestinal occludin localization (immunofluorescence microscopy). The human intestinal epithelial cell line Caco-2 was also analyzed in vitro for the effects of ethanol on the barrier function (transepithelial electrical resistance), occludin localization (immunofluorescence microscopy and Western blotting), and endocytosis pathways (double-labeling immunofluorescence microscopy with selective pathway inhibitors). The ethanol-fed mice developed steatohepatitis and displayed intestinal barrier dysfunction, the disruption of intestinal TJ, and enhanced intestinal endocytosis of occluding compared with the control mice. In the Caco-2 monolayers, ethanol treatment decreased transepithelial electrical resistance, disrupted TJ formation, and enhanced occludin endocytosis in a dose- and time-dependent manner. These deleterious events were reversed by pretreating the Caco-2 cells with a selective pharmacological inhibitor of macropinocytosis, but not with the inhibitors of clathrin or caveolin-mediated endocytic pathways. Chronic ethanol exposure may increase intestinal permeability by inducing the micropinocytosis of occludin, resulting in the disruption of intestinal TJ.

Macropinocytosis dependent entry of Chikungunya virus into human muscle cells.

Chikungunya virus (CHIKV) is a re-emerging arbovirus known to cause chronic myalgia and arthralgia with high morbidity. CHIKV is now considered endemic in many countries across Asia and Africa. In this study, the susceptibility of various human, mammalian and mosquito cell lines to CHIKV infection was evaluated. CHIKV infection was found to be cell-type dependent and virus strain-specific. Furthermore, SJCRH30 (human rhabdomyosarcoma cell line) was showed to be highly permissive to CHIKV infection, with maximum production of infectious virions observed at 12 h.p.i. Pre-infection treatment of SJCRH30 with various inhibitors of endocytosis, including monodansylcadaverine (receptor-mediated endocytic inhibitor), dynasore (clathrin-mediated endocytic inhibitor), as well as filipin (caveolin-mediated endocytosis inhibitor), resulted in minimal inhibition of CHIKV infection. In contrast, dose-dependent inhibition of CHIKV infection was observed with the treatment of macropinocytosis inhibitor, 5-(N-ethyl-N-isopropyl)amiloride (EIPA). Furthermore, siRNA-mediated knockdown of sortin nexin 9 (SNX9) a protein involved in macropinosome formation, also resulted in a significant dose-dependent reduction in viral titre. By performing a virus entry assay, CHIKV particles were also observed to colocalize with FITC-dextran, a macropinosome marker. This study shows for the first time, that the infectious entry of CHIKV into human muscle cells is mediated by macropinocytosis. Together, the data from this study may pave the way for the development of specific inhibitors that target the entry process of CHIKV into cells.

Abstract 279: c-Jun N-terminal kinase is involved in an oxidative stress- and vacuole-associated cell death pathway in triple negative breast cancer

Abstract Breast cancer is the most prevalent type of cancer among women. Triple negative breast cancer (TNBC) is a highly aggressive and heterogeneous subtype of breast cancer. It is the most challenging subtype to treat mainly due its aggressive nature and lack of targets such as HER2 (human epidermal growth factor 2), PR (progesterone receptor), and ER (estrogen receptor) found in other subtypes. We have shown that inhibiting both EGFR (epidermal growth factor receptor)/HER2 with lapatinib and JNK with JNK-IN-8 causes synergistic cell death in 11 out of 12 TNBC cell lines. EGFR is expressed in up to 90% tumors and JNK activation is linked to TNBC phenotype, metastasis, and stem cell character, among others. Synergism was achieved in the resistant, low-EGFR expressing cell line when a FGFR inhibitor (BGJ398) was used instead of lapatinib in combination with JNK-IN-8. Combination drug effects were accompanied by elevated oxidative stress. Suppression of oxidative stress restored cell viability in cells treated with the drug combination. JNK-IN-8 treatment alone or in combination with lapatinib induced massive cytoplasmic vacuolization with little apoptotic blebbing. The vacuoles were shown to be membrane-derived and acidic using Lucifer Yellow and Acridine Orange, respectively. Pre-treatment of cells with bafilomycin A1 (lysosome inhibitor), 5-(N-Ethyl-N-isopropyl) amiloride (macropinocytosis inhibitor), or N-acetylcysteine (antioxidant) reduced vacuolization. A similar effect was observed when cells were serum-starved. JNK1 knockout mouse mammary tumor cells, in comparison with JNK2 knockout, were resistant to drug synergism and displayed significantly less oxidative stress and vacuolization. Our studies demonstrated that concomitant blockade of JNK and individual tumor-appropriate targets induce synergistic cell death in TNBC cells in response to oxidative stress. Moreover, JNK blockade resulted in the formation of serum-dependent, acidic vacuoles that were reversible by lysosome or macropinocytosis inhibition. Loss-of-function studies indicated that JNK1 was the key isoform responsible for the drug response in TNBC cells. These findings suggest that JNK is involved in a novel cell death pathway and is a robust target in combination therapies against TNBC. Citation Format: Milad Soleimani, Ritvik Bhattacharjee, Uma Giri, Tamer S. Kaoud, Kevin N. Dalby, S. Gail Eckhardt, Carla L. Van Den Berg. c-Jun N-terminal kinase is involved in an oxidative stress- and vacuole-associated cell death pathway in triple negative breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 279.

Size-dependent cellular uptake and localization profiles of silver nanoparticles.

Purpose: Silver nanoparticles (AgNPs) have been widely applied in various fields as excellent antibacterial reagents over the past decades. Although the particle size is considered as the most crucial factor influencing cellular uptake, transportation, and accumulation behaviors, there are still many controversies regarding the correlation between size and uptake of AgNPs. In this study, size-dependent cellular uptake of AgNPs with different diameters was investigated in B16 cells.Methods: The uptake of AgNPs was investigated by inductively coupled plasma-mass spectrometry (ICP-MS) and transmission electron microscopic (TEM) imaging in B16 cells.Results: Twenty nanometer and 100 nm AgNPs had the lowest and highest uptake efficiency at both 12 hours and 24 hours, respectively. Smaller AgNPs crossed the plasma membrane faster with uniform distribution: 5 nm AgNPs were detected in both cytoplasm and nucleus at 0.5 hours after incubation. Larger AgNPs were extremely difficult to migrate: 100 nm AgNPs were detected in the nucleus at 12 hours after incubation. Internalization of AgNPs was directly observed, mainly within membrane-bound structures, such as intracellular vesicles and late endosomes. The uptake of all four-sized AgNPs (5 nm, 20 nm, 50 nm, 100 nm) decreased significantly after the pre-treatment with chlorpromazine hydrochloride, which can specifically inhibit the clathrin-mediated endocytosis. The internalization efficiencies of AgNPs (5 nm, 20 nm, 50 nm) were markedly reduced by methyl-β-cyclodextrin, a specific caveolin-mediated endocytosis inhibitor, whereas 5-(N-ethyl-N-isopropyl) amiloride as an inhibitor of macropinocytosis inhibited the uptake of larger sizes of AgNPs (50 nm and 100 nm).Conclusion: The results suggest that the size of AgNPs can not only affect the efficiency of cellular uptake, but also the type of endocytosis. The clathrin-mediated endocytosis may be the most common endocytic pathway for AgNPs in B16 cells, and AgNPs at each size were likely to enter cells by a major internalization pathway.

Inducible Membrane Permeabilization by Attenuated Lytic Peptides: A New Concept for Accessing Cell Interiors through Ruffled Membranes.

A variety of mid-sized and large biomolecules have been used as tools to explore fundamental biological questions. However, such molecules are often cell-impermeable and thus unable to attain sufficient access to the cell interior. This inhibits their ability to yield analytical data about the cell interior or modify the cellular events. We have recently developed a peptide, engineered from a natural hemolytic peptide, named L17E. Substantial cytosolic delivery of biomacromolecules, including antibodies, was attained in the presence of this peptide. In this study, detailed analysis of the modes of action of L17E was conducted, elucidating that a large fraction of the cytosolic translocation of biomacromolecules is accomplished in the presence of L17E within 5 min. L17E stimulates actin polymerization and induces a dynamic structural alteration of cell membranes, resulting in a ruffled appearance. Studies using macropinocytosis inhibitors and proteins that control endosome maturation raise the possibility that the transient permeabilization of ruffled cell membranes, rather than the rupture of endosomal membranes, is the crucial mechanism for facile cytosolic translocation of biomacromolecules in the presence of L17E. Our results provide a distinct concept of intracellular delivery, different from direct translocation through cell membranes or endocytic uptake followed by endosomal escape. This method of permeabilization via membrane ruffling provides a novel concept in intracellular delivery.

Energy-dependent endocytosis is responsible for drug transcorneal penetration following the instillation of ophthalmic formulations containing indomethacin nanoparticles.

PurposeWe previously found that ophthalmic formulations containing nanoparticles prepared by a bead mill method lead to an increase in bioavailability in comparison with traditional formulations (solution type). However, the transcorneal penetration pathway for ophthalmic formulations has not been explained yet. In this study, we investigated the mechanism of transcorneal penetration in the application of ophthalmic formulations containing indomethacin nanoparticles (IMC-NPs).Materials and methodsIMC-NPs was prepared by the bead mill method. For the analysis of energy-dependent endocytosis, corneal epithelial (HCE-T) cell monolayers and removed rabbit cornea were thermoregulated at 4°C, where energy-dependent endocytosis is inhibited. In addition, for the analysis of different endocytosis pathways using pharmacological inhibitors, inhibitors of caveolae-mediated endocytosis (54 µM nystatin), clathrin-mediated endocytosis (40 µM dynasore), macropinocytosis (2 µM rottlerin) or phagocytosis (10 µM cytochalasin D) were used.ResultsThe ophthalmic formulations containing 35–200 nm sized indomethacin nanoparticles were prepared by treatment with a bead mill, and no aggregation or degradation of indomethacin was observed in IMC-NPs. The transcorneal penetration of indomethacin was significantly decreased by the combination of nystatin, dynasore and rottlerin, and the decreased penetration levels were similar to those at 4°C in HCE-T cell monolayers and rabbit cornea. In the in vivo experiments using rabbits, dynasore and rottlerin tended to decrease the transcorneal penetration of indomethacin (area under the drug concentration – time curve in the aqueous humor [AUCAH]), and the AUCAH in the nystatin-treated rabbit was significantly lower than that in non-treatment group. In addition, the AUCAH in rabbit corneas undergoing multi-treatment was obviously lower than that in rabbit corneas treated with each individual endocytosis inhibitor.ConclusionWe found that three energy-dependent endocytosis pathways (clathrin-dependent endocytosis, caveolae-dependent endocytosis and macropinocytosis) are related to the trans-corneal penetration of indomethacin nanoparticles. In particular, the caveolae-dependent endocytosis is strongly involved.

Phellodendrine chloride suppresses proliferation of KRAS mutated pancreatic cancer cells through inhibition of nutrients uptake via macropinocytosis

Despite the massive efforts to develop the treatment of pancreatic cancers, no effective application exhibits satisfactory clinical outcome. Macropinocytosis plays a critical role for continuous proliferation of pancreatic ductal adenocarcinoma (PDAC). In this study, we generated a screening method and identified phellodendrine chloride (PC) as a potential macropinocytosis inhibitor. PC significantly inhibited the viability of KRAS mutant pancreatic cancer cells (PANC-1 and MiaPaCa-2) in a dose-dependent manner; however, it did not affect the wild type KRAS pancreatic cancer cells (BxPC-3). Further experiments indicated that PC reduced the growth of PANC-1 cells through inhibition of macropinocytosis and diminishing the intracellular glutamine level. Disruption of glutamine metabolism led to enhance the reactive oxygen species level and induce mitochondrial membrane potential depolarization in PANC-1 cells. PC treatment caused increased Bax and decreased Bcl-2 expression, along with the activation of cleaved caspase-3, 7, 9 and cleaved-PARP, thus induced mitochondrial apoptosis. Moreover, PC inhibited macropinocytosis in vivo and effectively reduced the growth of PANC-1 xenograft tumors. All together, we demonstrated that inhibition of macropinocytosis might be an effective strategy to treat pancreatic cancers. Thus, PC could be a potential compound with improved therapeutic efficacy in patients with pancreatic cancers.