Multifunctional chitosan-bimetallic nanocarrier deliver 5-fluorouracil for enhanced treatment of pancreatic and triple-negative breast cancer.

This work aimed to prepare multifunctional aptamer-conjugated, photothermally responsive 5-fluorouracil (5fu)-loaded chitosan-bimetallic (Au/Pd) nanoparticles (APT-CS-5fu-Au/Pd NPs) for improved cytotoxicity in two cancer cell lines (PANC-1 and MDA-MD 231). The CS-5fu-Au/Pd NPs were polydispersed with a size of 34.43 ± 1.59 nm. FTIR analysis indicated the presence of CS, 5fu in CS-5fu-Au/Pd NPs. The 2 theta degrees in CS-5fu-Au/Pd NPs accounted for CS and Au/Pd. Additionally, AGE revealed the conjugation of APT in CS-5fu-Au/Pd NPs. The APT-CS-5fu-Au/Pd NPs (180 µg/mL) with NIR treatment increased the temperature to >50 °C. The optimized 5fu input was 0.075 % in CS-5fu-Au/Pd NPs, exhibiting a hydrodynamic size of 112.96 ± 17.23 nm, DEE of 64.2 ± 3.77 %, and DLE of 11.1 ± 0.65 %. A higher level of 5fu release (69.8 ± 2.78 %) was observed under pH 5.4 at 74 h. In conclusion, NIR-APT-CS-5fu-Au/Pd NPs did not cause toxicity to RBC and Egg CAM, but increased cytotoxicity in MDA-MB 231 and PANC-1 cells by triggering oxidative stress-mediated cell death.

HDAC6-MYCN-CXCL3 axis mediates allergic inflammation and is necessary for allergic inflammation-promoted cellular interactions.

Histone deacetylase 6 (HDAC6) has been shown to play an important role in allergic inflammation. This study hypothesized that novel downstream targets of HDAC6 would mediate allergic inflammation. Experiments employing HDAC6 knock out C57BL/6 mice showed that HDAC6 mediated passive cutaneous anaphylaxis (PCA) and passive systemic anaphylaxis (PSA). Antigen stimulation increased expression of N-myc (MYCN) and CXCL3 in an HDAC6-dependent manner in the bone marrow-derived mast cells. MYCN and CXCL3 were necessary for both PCA and PSA. The role of early growth response 3 (EGR3) in the regulation of HDAC6 expression has been reported. ChIP assays showed EGR3 as a direct regulator of MYCN. miR-34a-5p was predicted to be a negative regulator of MYCN. Luciferase activity assays showed miR-34a-5p as a direct regulator of MYCN. miR-34a-5p mimic negatively regulated PCA and PSA. MYCN decreased miR-34a-5p expression in antigen-stimulated rat basophilic leukemia cells (RBL2H3). MYCN was shown to bind to the promoter sequence of CXCL3. In an IgE-independent manner, recombinant CXCL3 protein increased expression of HDAC6, MYCN, and ß-hexosaminidase activity in RBL2H3 cells. Mouse recombinant CXCL3 protein enhanced the angiogenic potential of the culture medium of RBL2H3. CXCL3 was necessary for the enhanced angiogenic potential of the culture medium of antigen-stimulated RBL2H3. The culture medium of RBL2H3 was able to induce M2 macrophage polarization in a CXCL3-dependent manner. Recombinant CXCL3 protein also increased the expression of markers of M2 macrophage. Thus, the identification of the novel role of HDAC6-MYCN-CXCL3 axis can help better understand the pathogenesis of anaphylaxis.

Cancer/testis antigen CAGE mediates osimertinib resistance in non-small cell lung cancer cells and predicts poor prognosis in patients with pulmonary adenocarcinoma.

CAGE, a cancer/testis antigen, was originally isolated from the sera of patients with gastric cancers. Previously, we have shown the role of CAGE in resistance to chemotherapy and target therapy. The aim of this study was to investigate the role of CAGE in osimertinib resistance and determine the prognostic value of CAGE in patients with pulmonary adenocarcinomas. The clinicopathological correlation with CAGE and autophagy flux in patients was examined using immunohistochemistry and in situ hybridization. The possible role of autophagy in osimertinib resistance was analyzed using immune blot, immune fluorescence staining and immunohistochemistry. This study found that immunohistochemical staining (IHC) showed CAGE expression in more than 50% of patients with pulmonary adenocarcinomas (pADCs). CAGE expression was increased in pADCs after the acquisition of EGFR-TKIs resistance. High expression of CAGE was correlated with shorter overall survival and progression free survival in patients with pADCs. Thus, CAGE mediates osimertinib resistance and predicts poor prognosis in patients with pADCs. Osimertinib-resistant non-small cell lung cancer cells (PC-9/OSI) were established and mechanistic studies of CAGE-mediated osimertinib resistance were performed. PC-9/OSI cells showed increased autophagic flux and CAGE expression compared with parental sensitive PC-9 cells. PC-9/OSI cells showed higher tumorigenic, metastatic, and angiogenic potential compared with parental PC-9 cells. CAGE CRISPR-Cas9 cell lines showed decreased autophagic flux, invasion, migration potential, and tumorigenic potential compared with PC-9/OSI cells in vitro and in vivo. CAGE plays a crucial role in the cancer progression by modulating autophagy and can predict the poor prognosis of patients with pulmonary adenocarcinomas. Our findings propose CAGE as a potential therapeutic target for developing anticancer drugs that can overcome osimertinib resistance.

Methionine consumption by cancer cells drives a progressive upregulation of PD-1 expression in CD4 T cells.

Programmed cell death protein 1 (PD-1), expressed on tumor-infiltrating T cells, is a T cell exhaustion marker. The mechanisms underlying PD-1 upregulation in CD4 T cells remain unknown. Here we develop nutrient-deprived media and a conditional knockout female mouse model to study the mechanism underlying PD-1 upregulation. Reduced methionine increases PD-1 expression on CD4 T cells. The genetic ablation of SLC43A2 in cancer cells restores methionine metabolism in CD4 T cells, increasing the intracellular levels of S-adenosylmethionine and yielding H3K79me2. Reduced H3K79me2 due to methionine deprivation downregulates AMPK, upregulates PD-1 expression and impairs antitumor immunity in CD4 T cells. Methionine supplementation restores H3K79 methylation and AMPK expression, lowering PD-1 levels. AMPK-deficient CD4 T cells exhibit increased endoplasmic reticulum stress and Xbp1s transcript levels. Our results demonstrate that AMPK is a methionine-dependent regulator of the epigenetic control of PD-1 expression in CD4 T cells, a metabolic checkpoint for CD4 T cell exhaustion.

Highly Excretable Gold Supraclusters for Translatable In Vivo Raman Imaging of Tumors.

Raman spectroscopy provides excellent specificity for in vivo preclinical imaging through a readout of fingerprint-like spectra. To achieve sufficient sensitivity for in vivo Raman imaging, metallic gold nanoparticles larger than 10 nm were employed to amplify Raman signals via surface-enhanced Raman scattering (SERS). However, the inability to excrete such large gold nanoparticles has restricted the translation of Raman imaging. Here we present Raman-active metallic gold supraclusters that are biodegradable and excretable as nanoclusters. Although the small size of the gold nanocluster building blocks compromises the electromagnetic field enhancement effect, the supraclusters exhibit bright and prominent Raman scattering comparable to that of large gold nanoparticle-based SERS nanotags due to high loading of NIR-resonant Raman dyes and much suppressed fluorescence background by metallic supraclusters. The bright Raman scattering of the supraclusters was pH-responsive, and we successfully performed in vivo Raman imaging of acidic tumors in mice. Furthermore, in contrast to large gold nanoparticles that remain in the liver and spleen over 4 months, the supraclusters dissociated into small nanoclusters, and 73% of the administered dose to mice was excreted during the same period. The highly excretable Raman supraclusters demonstrated here offer great potential for clinical applications of in vivo Raman imaging.

Photothermally responsive chitosan-coated iron oxide nanoparticles for enhanced eradication of bacterial biofilms.

This work developed a pH/NIR responsive antibacterial agent (CS-FeNPs) composed of chitosan (CS) and Fe3O4 nanoparticles (FeNPs). CS triggers bacterial attraction through surface charge, while Fe acts as a photothermal agent (PTA). The CS-Fe NPs exhibited antibacterial and antibiofilm activity against both bacteria (G+/G-). However, higher activity was observed against bacteria (G-) due to electrostatic interactions. The CS-FeNPs bind with the bacterial membrane through electrostatic interactions and disturb bacterial cells. Later, in an acidic environment, CS-FeNPs bind with bacterial membrane, and NIR irradiation leads the antibacterial activity. CS-FeNPs exhibited a potential photothermal conversion efficiency (η) of 21.53 %. Thus, it converts NIR irradiation into heat to kill the bacterial pathogen. The CS-FeNPs were found to be less cytotoxic with great antibacterial efficiency on planktonic bacteria and their biofilm, which indicates that they deserve to develop potential and safe treatment strategies for the treatment of bacterial infections.

Obesity Exacerbates Coxsackievirus Infection via Lipid-Induced Mitochondrial Reactive Oxygen Species Generation.

Coxsackievirus B3 (CVB3) infection causes acute pancreatitis and myocarditis. However, its pathophysiological mechanism is unclear. Here, we investigated how lipid metabolism is associated with exacerbation of CVB3 pathology using high-fat diet (HFD)-induced obese mice. Mice were intraperitoneally inoculated with 1×106 pfu/mouse of CVB3 after being fed a control or HFD to induce obesity. Mice were treated with mitoquinone (MitoQ) to reduce the level of mitochondrial ROS (mtROS). In obese mice, lipotoxicity of white adipose tissue-induced inflammation caused increased replication of CVB3 and mortality. The coxsackievirus adenovirus receptor increased under obese conditions, facilitating CVB3 replication in vitro. However, lipid-treated cells with receptor-specific inhibitors did not reduce CVB3 replication. In addition, lipid treatment increased mitochondria-derived vesicle formation and the number of multivesicular bodies. Alternatively, we found that inhibition of lipid-induced mtROS decreased viral replication. Notably, HFD-fed mice were more susceptible to CVB3-induced mortality in association with increased levels of CVB3 replication in adipose tissue, which was ameliorated by administration of the mtROS inhibitor, MitoQ. These results suggest that mtROS inhibitors can be used as potential treatments for CVB3 infection.

Esculetin and Fucoidan Attenuate Autophagy and Apoptosis Induced by Zinc Oxide Nanoparticles through Modulating Reactive Astrocyte and Proinflammatory Cytokines in the Rat Brain.

We examined the protective effects of esculetin and fucoidan against the neurotoxicity of ZnO NPs in rats. Ninety rats were divided into nine groups and pre-treated with esculetin or fucoidan 1 h before ZnO NP administration on a daily basis for 2 weeks. Serum and brain homogenates were examined by enzyme-linked immunosorbent assay (ELISA), and neurons, microglia, and astrocytes in the hippocampal region were examined with immunohistochemical analysis. The serum levels of interleukin-1-beta (IL-1ß), 3-nitrotyrosine (3-NT), superoxide dismutase (SOD), and 8-hydroxy-2'-deoxyguanosine (8-OHdG) were altered in the ZnO NP treatment groups. Brain IL-1ß and TNF-α levels were elevated after ZnO NP administration, and these effects were inhibited by esculetin and fucoidan. SOD, 8-OHdG, and acetylcholinesterase (AChE) levels in the brain were decreased after ZnO NP administration. The brain levels of beclin-1 and caspase-3 were elevated after ZnO NP treatment, and these effects were significantly ameliorated by esculetin and fucoidan. The number of reactive astrocytes measured by counting glial fibrillary acidic protein (GFAP)-positive cells, but not microglia, increased following ZnO NP treatment, and esculetin and fucoidan ameliorated the changes. Esculetin and fucoidan may be beneficial for preventing ZnO NP-mediated autophagy and apoptosis by the modulation of reactive astrocyte and proinflammatory cytokines in the rat brain.

Folic acid conjugated chitosan encapsulated palladium nanoclusters for NIR triggered photothermal breast cancer treatment.

This study developed folic acid (FA) conjugated chitosan (CS) encapsulated rutin (R) synthesized palladium nanoclusters (Pd NCs) for NIR triggered and folate receptor (FR) targeted triple-negative breast cancer (MDA-MB 231 cells) treatment. R-Pd NCs exhibited flower-shaped particles with an average size of <100 nm. FA-CS encapsulation concealed the flower shape of R-Pd NCs with a positive charge. The XRD spectrum confirmed the cubic crystalline structure of Pd. The FA conjugation on CS improved the cellular uptake of R-Pd NCs in MDA-MB 231 cells was confirmed by TEM. FA-CS-R-Pd NCs (+NIR) treatment was considerably inhibited the MDA-MB 231 cells proliferation evidenced by cell viability, fluorescent staining, and flow cytometry analysis. Further, in vitro hemolysis assay and in Ovo model confirmed the non-toxic nature of FA-CS-R-Pd-NCs with or without NIR radiation. Hence, this study concluded that FA-CS-R-Pd NCs can be applied for the treatment of drug-resistant breast cancer.

Inhibitors of Lipoxygenase and Cyclooxygenase-2 Attenuate Trimethyltin-Induced Neurotoxicity through Regulating Oxidative Stress and Pro-Inflammatory Cytokines in Human Neuroblastoma SH-SY5Y Cells.

Trimethyltin (TMT) is an environmental neurotoxin that mediates dopaminergic neuronal injury in the brain. In this study, we characterized the toxic mechanism and possible protective compounds against TMT-induced neurotoxicity in human dopaminergic neuroblastoma SH-SY5Y cells. Antioxidants such as melatonin, N-acetylcysteine (NAC), α-tocopherol, and allopurinol alleviated TMT toxicity. Apoptosis induced by TMT was identified by altered expression of cleaved caspase-3, Bax, Bcl-2, and Bcl-xL through Western blot analysis. The iron chelator deferoxamine ameliorated the alteration of apoptosis-related proteins through TMT exposure. TMT also induced delayed ultrastructural necrotic features such as mitochondrial swelling and cytoplasmic membrane rupture; NAC reduced these necrotic injuries. Esculetin, meloxicam, celecoxib, and phenidone decreased TMT toxicity. Elevation of the pro-inflammatory cytokines IL-1ß, TNF-α, and NF-ĸB and reduction of the antioxidant enzymes catalase and glutathione peroxidase-1 (GPx-1) were induced by TMT and ameliorated by inhibitors of LOX and COX-2 enzymes. Both NMDA and non-NMDA antagonists attenuated TMT toxicity. The free calcium ion modulators nimodipine and BAPTA/AM contributed to neuronal survival against TMT toxicity. Inhibitors of the phosphoinositide 3-kinase/protein kinase B/mammalian target of rapamycin pathway, an autophagy regulator, decreased TMT toxicity. These results imply that TMT neurotoxicity is the chief participant in LOX- and COX-2-mediated apoptosis, partly via necrosis and autophagy in SH-SY5Y cells.

EGR3-HDAC6-IL-27 Axis Mediates Allergic Inflammation and Is Necessary for Tumorigenic Potential of Cancer Cells Enhanced by Allergic Inflammation-Promoted Cellular Interactions.

The objective of this study was to investigate mechanisms of allergic inflammation both in vitro and in vivo in details. For this, RNA sequencing was performed. Early growth response 3 gene (Egr3) was one of the most highly upregulated genes in rat basophilic leukemia (RBL2H3) cells stimulated by antigen. The role of Egr3 in allergic inflammation has not been studied extensively. Egr3 was necessary for passive cutaneous anaphylaxis (PCA) and passive systemic anaphylaxis (PSA). Egr3 promoter sequences contained potential binding site for NF-κB p65. NF-κB p65 directly regulated Egr3 expression and mediated allergic inflammation in vitro. Histone deacetylases (HDACs) is known to be involved in allergic airway inflammation. HDAC6 promoter sequences contained potential binding site for EGR3. EGR3 showed binding to promoter sequences of HDAC6. EGR3 was necessary for increased expression of histone deacetylase 6 (HDAC6) in antigen-stimulated RBL2H3 cells. HDAC6 mediated allergic inflammation in vitro and PSA. TargetScan analysis predicted that miR-182-5p was a negative regulator of EGR3. Luciferase activity assay confirmed that miR-182-5p was a direct regulator of EGR3. MiR-182-5p mimic inhibited allergic inflammation both in vitro and in vivo. Cytokine array showed that HDAC6 was necessary for increased interleukin-27 (IL-27) expression in BALB/C mouse model of PSA. Antigen stimulation did not affect expression of EBI3, another subunit of IL-27 in RBL2H3 cells or BALB/C mouse model of PCA or PSA. IL-27 receptor alpha was shown to be able to bind to HDAC6. IL-27 p28 mediated allergic inflammation in vitro, PCA, and PSA. Mouse recombinant IL-27 protein promoted features of allergic inflammation in an antigen-independent manner. HDAC6 was necessary for tumorigenic and metastatic potential enhanced by PSA. PSA enhanced the metastatic potential of mouse melanoma B16F1 cells in an IL-27-dependent manner. Experiments employing culture medium and mouse recombinant IL-27 protein showed that IL-27 mediated and promoted cellular interactions involving B16F1 cells, lung macrophages, and mast cells during allergic inflammation. IL-27 was present in exosomes of antigen-stimulated RBL2H3 cells. Exosomes from antigen-stimulated RBL2H3 cells enhanced invasion of B16F1 melanoma cells in an IL-27-dependemt manner. These results present evidence that EGR3-HDAC6-IL-27 axis can regulate allergic inflammation by mediating cellular interactions.

MiR-154-5p-MCP1 Axis Regulates Allergic Inflammation by Mediating Cellular Interactions.

In a previous study, we have demonstrated that p62, a selective receptor of autophagy, can regulate allergic inflammation. In the present study, microRNA array analysis showed that miR-154-5p was increased by antigen (DNP-HSA) in a p62-dependent manner in rat basophilic leukemia cells (RBL2H3). NF-kB directly increased the expression of miR-154-5p. miR-154-5p mediated in vivo allergic reactions, including passive cutaneous anaphylaxis and passive systemic anaphylaxis. Cytokine array analysis showed that antigen stimulation increased the expression of MCP1 in RBL2H3 cells in an miR-154-5p-dependent manner. Reactive oxygen species (ROS)-ERK-NF-kB signaling increased the expression of MCP1 in antigen-stimulated RBL2H3 cells. Recombinant MCP1 protein induced molecular features of allergic reactions both in vitro and in vivo. Anaphylaxis-promoted tumorigenic potential has been known to be accompanied by cellular interactions involving mast cells, and macrophages, and cancer cells. Our experiments employing culture medium, co-cultures, and recombinant MCP1 protein showed that miR-154 and MCP1 mediated these cellular interactions. MiR-154-5p and MCP1 were found to be present in exosomes of RBL2H3 cells. Exosomes from PSA-activated BALB/C mouse induced molecular features of passive cutaneous anaphylaxis in an miR-154-5p-dependent manner. Exosomes from antigen-stimulated RBL2H3 cells enhanced both tumorigenic and metastatic potentials of B16F1 melanoma cells in an miR-154-5p-dependent manner. Exosomes regulated both ROS level and ROS mediated cellular interactions during allergic inflammation. Our results indicate that the miR-154-5p-MCP1 axis might serve as a valuable target for the development of anti-allergy therapeutics.

The CAGE-MiR-181b-5p-S1PR1 Axis Regulates Anticancer Drug Resistance and Autophagy in Gastric Cancer Cells.

Cancer-associated gene (CAGE), a cancer/testis antigen, has been known to promote anticancer drug resistance. Since the underlying mechanisms of CAGE-promoted anticancer drug resistance are poorly understood, we established Anticancer drug-resistant gastric cancer cells (AGS R ) to better elucidate possible mechanisms. AGS R showed an increased expression level of CAGE and autophagic flux compared with anticancer drug-sensitive parental gastric cancer cells (AGS cells). AGS R cells showed higher invasion potential, growth rate, tumor spheroid formation, and angiogenic potential than AGS cells. CAGE exerted effects on the response to anticancer drugs and autophagic flux. CAGE was shown to bind to Beclin1, a mediator of autophagy. Overexpression of CAGE increased autophagic flux and invasion potential but inhibited the cleavage of PARP in response to anticancer drugs in CAGE CRISPR-Cas9 cell lines. TargetScan analysis was utilized to predict the binding of miR-302b-5p to the promoter sequences of CAGE, and the results show that miR-302b-5p directly regulated CAGE expression as illustrated by luciferase activity. MiR-302b-5p regulated autophagic flux and the response to anticancer drugs. CAGE was shown to bind the promoter sequences of miR-302b-5p. The culture medium of AGS R cells increased CAGE expression and autophagic flux in AGS cells. ImmunoEM showed CAGE was present in the exosomes of AGS R cells; exosomes of AGS R cells and human recombinant CAGE protein increased CAGE expression, autophagic flux, and resistance to anticancer drugs in AGS cells. MicroRNA array revealed miR-181b-5p as a potential negative regulator of CAGE. MiR-181b-5p inhibitor increased the expression of CAGE and autophagic flux in addition to preventing anticancer drugs from cleaving poly(ADP-ribose) polymerase (PARP) in AGS cells. TargetScan analysis predicted sphingosine 1-phosphate receptor 1 (SIPR1) as a potential target for miR-181b-5p. CAGE showed binding to the promoter sequences of S1PR1. The downregulation or inhibition of S1PR1 led to decreased autophagic flux but enhanced the sensitivity to anticancer drugs in AGS R cells. This study presents a novel role of the CAGE-miR-181b-5p-S1PR1 axis in anticancer drug resistance and autophagy.

1-Cinnamoyltrichilinin from Melia azedarach Causes Apoptosis through the p38 MAPK Pathway in HL-60 Human Leukemia Cells.

Acute myeloid leukemia (AML) is an aggressive type of human leukemia with a low survival rate, and its complete remission remains challenging. Although chemotherapy is the first-line treatment of AML, it exerts toxicity in noncancerous cells when used in high doses, thus necessitating the development of novel compounds with a high therapeutic window. This study aimed to investigate the anticancer effects of several compounds derived from the fruits of Melia azedarach (a tree with medicinal properties). Among them, 1-cinnamoyltrichilinin (CT) was found to strongly suppress the viability of HL-60 human leukemia cells. CT treatment induced apoptosis and increased nuclear fragmentation and fractional DNA content in HL-60 cells in a dose-dependent manner. CT induced phosphorylation of p38 mitogen-activated protein kinases (p38), though not of c-Jun N-terminal kinase (JNK) and extracellular signal-regulated kinase (ERK), and activated Bcl-2 family proteins towards the proapoptosis and cleavage of caspase-3 and poly (ADP-ribose) polymerase. Both CT-mediated apoptosis and apoptotic protein expression were reversed by treatment with the p38 inhibitor, thereby indicating the p38 pathway to be critical in CT-stimulated apoptosis. The results collectively indicated CT to suppress HL-60 survival by activating the p38 pathway and inducing apoptosis, hence being a novel potential therapeutic agent for AML.

Hyaluronic Acid-Decorated Glycol Chitosan Nanoparticles for pH-Sensitive Controlled Release of Doxorubicin and Celecoxib in Nonsmall Cell Lung Cancer.

Nonsmall cell lung cancer (NSCLC) is a leading cause of global cancer mortality. Recently, combinatorial treatment approaches have shown promise as they better address tumor heterogeneity. However, drug pharmacokinetics and tissue distribution differences remain problematic. To overcome these issues and improve therapeutic efficacies, the use of nanomedicines has been suggested. We devised a CD44 receptor target hyaluronic acid (HA)-decorated glycol chitosan (GC) nanoparticle which is conjugated to doxorubicin (DOX) by a pH-sensitive linker and coloaded celecoxib (CXB; HA-GC-DOX/CXB). Successful chemical conjugation of GC to DOX was confirmed and HA-GC-DOX/CXB showed ∼150 nm of uniform spherical shape. HA-GC-DOX/CXB were stable at pH 7.4 but steadily increased in size and released drugs at pH 6.0 and 4.0. In vitro NSCLC cells showed that DOX and CXB combination therapy has synergism in both free drug and nanoparticle formulation. In vivo NSCLC xenograft mice showed DOX and CXB exhibited a synergistic tumor suppressive effect in HA-GC-DOX/CXB. Furthermore, HA-GC-DOX/CXB dramatically inhibited tumor growth compared to other treatments as well as suppressed inflammation and metastasis-related gene/protein in the tumor tissues. Our findings demonstrate HA-GC-DOX/CXB is a potential anticancer therapy that controlled release under acidic tumor microenvironments and enhanced CD44 overexpressed tumor target efficacy.

MiR-135-5p-p62 Axis Regulates Autophagic Flux, Tumorigenic Potential, and Cellular Interactions Mediated by Extracellular Vesicles During Allergic Inflammation.

The objective of this study was to investigate the relationship between autophagy and allergic inflammation. In vitro allergic inflammation was accompanied by an increased autophagic flux in rat basophilic leukemia (RBL2H3) cells. 3-MA, an inhibitor of autophagic processes, negatively regulated allergic inflammation both in vitro and in vivo. The role of p62, a selective receptor of autophagy, in allergic inflammation was investigated. P62, increased by antigen stimulation, mediated in vitro allergic inflammation, passive cutaneous anaphylaxis (PCA), and passive systemic anaphylaxis (PSA). P62 mediated cellular interactions during allergic inflammation. It also mediated tumorigenic and metastatic potential of cancer cells enhanced by PSA. TargetScan analysis predicted that miR-135-5p was a negative regulator of p62. Luciferase activity assay showed that miR-135-5p directly regulated p62. MiR-135-5p mimic negatively regulated features of allergic inflammation and inhibited tumorigenic and metastatic potential of cancer cells enhanced by PSA. MiR-135-5p mimic also inhibited cellular interactions during allergic inflammation. Extracellular vesicles mediated allergic inflammation both in vitro and in vivo. Extracellular vesicles were also necessary for cellular interactions during allergic inflammation. Transmission electron microscopy showed p62 within extracellular vesicles of antigen-stimulated rat basophilic leukemia cells (RBL2H3). Extracellular vesicles isolated from antigen-stimulated RBL2H3 cells induced activation of macrophages and enhanced invasion and migration potential of B16F1 mouse melanoma cells in a p62-dependent manner. Extracellular vesicles isolated from PSA-activated BALB/C mouse enhanced invasion and migration potential of B16F1 cells, and induced features of allergic inflammation in RBL2H3 cells. Thus, miR-135-5p-p62 axis might serve as a target for developing anti-allergy drugs.

Sulfisoxazole inhibits the secretion of small extracellular vesicles by targeting the endothelin receptor A.

Inhibitors of the secretion of cancer exosomes, which promote cancer progression and metastasis, may not only accelerate exosome biology research but also offer therapeutic benefits for cancer patients. Here we identify sulfisoxazole (SFX) as an inhibitor of small extracellular vesicles (sEV) secretion from breast cancer cells through interference with endothelin receptor A (ETA). SFX, an FDA-approved oral antibiotic, showed significant anti-tumor and anti-metastatic effects in mouse models of breast cancer xenografts, the reduced expression of proteins involved in biogenesis and secretion of sEV, and triggered co-localization of multivesicular endosomes with lysosomes for degradation. We demonstrate the important role of ETA, as target of SFX, by gain- and loss-of-function studies of the ETA protein, through a direct binding assay, and pharmacological and genetic approaches. These findings may provide a foundation for sEV-targeted cancer therapies and the mechanistic studies on sEV biology.

Development of a novel short 12-meric papiliocin-derived peptide that is effective against Gram-negative sepsis.

The development of novel peptide antibiotics with potent activity against multidrug-resistant Gram-negative bacteria and anti-septic activity is urgently needed. In this study, we designed short, 12-meric antimicrobial peptides by substituting amino acids from the N-terminal 12 residues of the papiliocin (Pap12-1) peptide to alter cationicity and amphipathicity and improve antibacterial activity and bacterial membrane interactions. Pap12-6, with an amphipathic α-helical structure and Trp12 at the C-terminus, showed broad-spectrum antibacterial activity, especially against multidrug-resistant Gram-negative bacteria. Dye leakage, membrane depolarization, and electron microscopy data proved that Pap12-6 kills bacteria by permeabilizing the bacterial membrane. Additionally, Pap12-6 significantly reduced the secretion of NO, TNF-α, and IL-6 and secreted alkaline phosphatase reporter gene activity confirmed that Pap12-6 shows anti-inflammatory activity via a TLR4-mediated NF-κB signaling pathway. In a mouse sepsis model, Pap12-6 significantly improved survival, reduced bacterial growth in organs, and reduced LPS and inflammatory cytokine levels in the serum and organs. Pap12-6 showed minimal cytotoxicity towards mammalian cells and controlled liver and kidney damage, proving its high bacterial selectivity. Our results suggest that Pap12-6 is a promising peptide antibiotic for the therapeutic treatment of Gram-negative sepsis via dual bactericidal and immunomodulatory effects on the host.

The roles of NF-κB and ROS in regulation of pro-inflammatory mediators of inflammation induction in LPS-stimulated zebrafish embryos.

In this study, the roles of reactive oxygen species (ROS) and NF-κB on inflammation induction in lipopolysaccharide (LPS)-stimulated zebrafish embryos were evaluated using N-acetyl-l-cysteine (NAC) and pyrrolidine dithiocarbamate (PDTC), specific inhibitors of ROS and NF-κB, respectively. LPS-stimulated zebrafish embryos showed increasing production of NO and ROS and expression of iNOS and COX-2 protein, compared to a control group without LPS. However, NAC significantly inhibited production of NO and ROS and markedly suppressed expression of iNOS and COX-2 protein in LPS-stimulated zebrafish embryos. The mRNA expressions of NF-κB such as p65NF-κB and IκB-A were significantly increased after LPS stimulation, whereas PDTC attenuated mRNA expression of NF-κB. PDTC also inhibited production of NO and reduced expression of iNOS and COX-2 protein in LPS-stimulated zebrafish embryos. Taken together, these results indicated that LPS increases pro-inflammatory mediators in zebrafish embryos through ROS and NF-κB regulation.

In vitro photodynamic effects of scavenger receptor targeted-photoactivatable nanoagents on activated macrophages.

Scavenger receptors (SRs) expressed on the activated macrophages in inflammation sites have been considered as the most interesting and important target biomarker for targeted drug delivery, imaging and therapy. In the present study, we fabricated the scavenger receptor-A (SR-A) targeted-photoactivatable nanoagents (termed as Ce6/DS-DOCA) by entrapping chlorin e6 (Ce6) into the amphiphilic dextran sulfate-deoxycholic acid (DS-DOCA) conjugates via physically hydrophobic interactions. Insoluble Ce6 was easily encapsulated into DS-DOCA nanoparticles by a dialysis method and the loading efficiency was approximately 51.7%. The Ce6/DS-DOCA formed nano-sized self-assembled aggregates (28.8±5.6nm in diameter), confirmed by transmission electron microscope, UV/Vis and fluorescence spectrophotometer. The Ce6/DS-DOCA nanoagents could generate highly reactive singlet oxygen under laser irradiation. Also, in vitro studies showed that they were more specifically taken up by lipopolysaccharide (LPS)-induced activated macrophages (RAW 264.7) via a SR-A-mediated endocytosis, relative to by non-activated macrophages, and notably induced cell death of activated macrophages under laser irradiation. Therefore, SR-A targetable and photoactivatable Ce6/DS-DOCA nanoagents with more selective targeting to the activated macrophages will have great potential for treatment of inflammatory diseases.