Cationic Polyphosphazene Vesicles for Cancer Immunotherapy by Efficient in Vivo Cytokine IL-12 Plasmid Delivery

To circumvent the severe toxicity of the systemic delivery of IL-12 protein and the limits of local administration of IL-12 gene, we constructed a polymersome system for systemic delivery of recombinant murine IL-12 plasmid (pmIL-12) based on amphiphilic polyphosphazenes containing weakly cationic N,N-diisopropylethylene­diamine (DPA) as hydrophobic groups and monomethoxy poly­(ethylene glycol) (mPEG) as hydrophilic tails. By simple dialysis method, pmIL-12 was successfully loaded into polymersomes due to the combination effect of physical encapsulation and electrostatic interaction. This pmIL-12 polymersome delivery system was validated with good biocompatibility and stability despite of serum protein and DNase challenging. The results of in vivo antitumor experiments showed that intravenous injection of pmIL-12 polymersomes achieved significant suppression of tumor growth in BALB/c mice bearing CT-26 colon carcinoma. The analysis revealed that the mechanism was related to the antitumor immune response induced by efficient transfection of pmIL-12 polymersomes, which maybe involved lymphocytes infiltration and angiogenic inhibition at the tumor site

Mast cells disrupt the duodenal mucosal integrity: Implications for the mechanisms of barrier dysfunction in functional dyspepsia

Functional dyspepsia (FD) is a common functional gastrointestinal (GI) disorder, but its pathophysiology is poorly understood. Mast cells (MCs) may play a critical role in the development of FD. Therefore, the aim of this study was to investigate the effect of MCs on barrier function, tight junction (TJ) proteins and related signaling pathways. The expression of the TJ proteins claudin-8, ZO-1 and occludin in biopsy tissues from seven FD patients and five controls was assessed. Based on the in vivo results, we further investigated the effect of (1) MC degranulation in a coculture model of Caco-2/RBL-2H3 cells and tryptase in Caco-2 monolayers, (2) MC degranulation in the presence or absence of a PAR-2 antagonist and (3) MC degranulation in the presence or absence of an ERK1/2 signaling pathway inhibitor. The epithelial integrity of Caco-2 cell monolayers was assessed by measuring the transepithelial electrical resistance (TEER). The expression of TJ proteins was evaluated by western blotting, QT-PCR and immunostaining. Epithelial claudin-8, ZO-1 and occludin protein expression were significantly reduced in tissues from FD patients compared with controls. MC degranulation and tryptase decreased the TEER and reduced the expression of TJ proteins in Caco-2 cell monolayers. A PAR-2 antagonist and an ERK1/2 signaling pathway inhibitor significantly reduced the effect of MC degranulation on the TEER and TJ protein expression in Caco-2 cell monolayers. MCs disrupt duodenal barrier function by modulating the levels of TJ proteins, and the PAR-2 and ERK1/2 signaling pathways may mediate the pathogenesis of FD.</p

STAT1 knockdown blocks IFNγ-induced IL-6 and IL-8 in monolayer HEECs.

<p>STAT1 siRNA and non-specific control siRNA (negative siRNA) were transfected into monolayer HEECs 72 h before stimulation. (<i>A</i>) STAT1 expression was evaluated using RT-qPCR after transfection. (<i>B</i>) STAT1 production was evaluated by western blot analysis after transfection. (<i>C</i>, <i>D</i>) The production of IL-6 (<i>C</i>) and IL-8 (<i>D</i>) 24 h after IFNγ (30 ng/ml) stimulation was detected by ELISA in the supernatants of negative siRNA and IL-33 siRNA treated groups. Each value represents the mean ± SD of 3 independent experiments. **<i>P</i> < 0.01.</p

Farnesylthiosalicylic acid sensitizes hepatocarcinoma cells to artemisinin derivatives

<div><p>Dihydroartemisinin (DHA) and artesunate (ARS), two artemisinin derivatives, have efficacious anticancer activities against human hepatocarcinoma (HCC) cells. This study aims to study the anticancer action of the combination treatment of DHA/ARS and farnesylthiosalicylic acid (FTS), a Ras inhibitor, in HCC cells (Huh-7 and HepG2 cell lines). FTS pretreatment significantly enhanced DHA/ARS-induced phosphatidylserine (PS) externalization, Bak/Bax activation, mitochondrial membrane depolarization, cytochrome <i>c</i> release, and caspase-8 and -9 activations, characteristics of the extrinsic and intrinsic apoptosis. Pretreatment with Z-IETD-FMK (caspase-8 inhibitor) potently prevented the cytotoxicity of the combination treatment of DHA/ARS and FTS, and pretreatment with Z-VAD-FMK (pan-caspase inhibitor) significantly inhibited the loss of ΔΨm induced by DHA/ARS treatment or the combination treatment of DHA/ARS and FTS in HCC cells. Furthermore, silencing Bak/Bax modestly but significantly inhibited the cytotoxicity of the combination treatment of DHA/ARS and FTS. Interestingly, pretreatment with an antioxidant N-Acetyle-Cysteine (NAC) significantly prevented the cytotoxicity of the combination treatment of DHA and FTS instead of the combination treatment of ARS and FTS, suggesting that reactive oxygen species (ROS) played a key role in the anticancer action of the combination treatment of DHA and FTS. Similar to FTS, DHA/ARS also significantly prevented Ras activation. Collectively, our data demonstrate that FTS potently sensitizes Huh-7 and HepG2 cells to artemisinin derivatives via accelerating the extrinsic and intrinsic apoptotic pathways.</p></div

Data_Sheet_1_FTX271: A potential gene resource for plant antiviral transgenic breeding.docx

Flammutoxin (FTX), as well as its precursor TDP, is a protein from Flammulina velutipes with antiviral activity. Transgenic tobacco with the FTX271 (gene of FTX or TDP) can not only delay the onset time of symptoms but also alleviate the symptoms caused by tobacco mosaic virus (TMV), but the mechanism is still unclear. In this study, FTX271 was introduced into Nicotiana benthamiana, and the disease resistance mechanism activated by FTX271 was speculated by transcriptomic and proteomic techniques. The results showed that TDP was detected, and some genes, proteins and pathways were significant upregulated or enriched in transgenic tobacco, including the mitogen-activated protein kinase (MAPK) cascade signal transduction pathway, the expression of hypersensitive response (HR) marker genes H1N1 and HSR203J, pathogenesis-related (PR) genes, and the key genes COI1 and lipoxygenase gene LOX2 of the jasmonic acid (JA) signaling pathway, indicating FTX271 may activate the MAPK pathway and increase the content of reactive oxygen species (ROS) and JA, which promoted the HR and inducible systemic resistance (ISR). ISR caused increased expression of peroxidase (POD) and other proteins involved in pathogen defense. In addition, transgenic tobacco may use sHSP-assisted photoreparation to alleviate the symptoms of TMV. In conclusion, JA-mediated ISR and sHSP-assisted photoreparation are activated by FTX271 to protect tobacco from TMV infection and alleviate the symptoms caused by the virus. The study provided a theoretical basis for the TMV resistance mechanism of FTX271, which may represent a potential gene resource for plant antiviral transgenic breeding.</p

DHA/ARS inhibits Ras activation.

<p>(<b>A</b>) FRET efficiency images of HepG2 cells expressing Raichu-Ras plasmid quantified by PbFRET quantification method. D: fluorescence intensities images from donor channel (Emission 480/40 nm) during donor excitation (Excitation 435/20 nm); A: fluorescence intensities images from acceptor channel (Emission 550/40 nm) during acceptor excitation (Excitation 510/17 nm); DP: fluorescence intensities images from donor channel during donor excitation after photobleaching (Excitation 510/17 nm); AP: fluorescence intensities images from acceptor channel during acceptor excitation after photobleaching (Excitation 510/17 nm). Scale bar: 10 μm. (<b>B</b>) Statistical results of FRET efficiency from at least 90 living HepG2 cells expressing Raichu-Ras plasmid after different treatments. <i>NS</i> = no statistical significance, <i>P</i> > 0.05; *<i>P</i> < 0.05, compared with control from the group treated with FBS for 2 h; ^#<i>P</i> < 0.05, compared with control from the group treated with FBS for 24 h.</p

IFNγ, but not TNF-α, upregulates nuclear IL-33.

<p>(<i>A</i>, <i>B</i>) IL-33 mRNA was analyzed by RT-qPCR. (<i>A</i>) ALI-cultured HEECs were stimulated with IFNγ (30 ng/ml), TNF-α (20 ng/ml), or both from the basal compartment, and harvested after the indicated time points. (<i>B</i>) ALI-cultured HEECs were stimulated with IFNγ (0.1, 1, 5, and 30 ng/ml) and harvested after 6 h. Each value represents the mean ± SD of 3 independent experiments. *<i>P</i> < 0.05, **<i>P</i> < 0.01. (<i>C</i>) Immunofluorescence staining of IL-33 (red) in ALI-cultured HEECs after IFNγ (30 ng/ml) stimulation at the indicated time points was performed. DAPI (blue) was used as the nuclear marker. Bar = 50 μm. (<i>D</i>) Relative levels of IL-33 in total or nuclear protein extract were assessed by western blot analysis at the indicated time points after IFNγ (30 ng/ml) stimulation.</p

Synergistic cytotoxicity of the combination treatment of DHA/ARS and FTS in Huh-7 cells.

<p>Synergistic cytotoxicity of the combination treatment of DHA/ARS and FTS in Huh-7 cells.</p

IFNγ-induced cytokine production is p38 MAPK and JAK/STAT1 dependent.

<p>ALI-cultured HEECs were pre-incubated with inhibitors of JAKs (2 μM, JAKi), p38 MAPK (40 μM, SB), PKA (10 μM, H89), or STAT1 (20 μM, EGCG) for 1 h, and subsequently co-incubated with IFNγ (30 ng/ml). (<i>A</i>) Cells were harvested 10 h after IFNγ stimulation to evaluate IL-33 production by western blot analysis (loading control: β-actin). (<i>B</i>) Media from the basal compartment were harvested 24 h after IFNγ stimulation to analyze IL-6, IL-8, RANTES, and MCP-1 production using the Bio-Plex assay. Each value represents the mean ± SD of 3 independent experiments. **<i>P</i> < 0.01 vs. Control. ^## <i>P</i> < 0.01 vs. IFNγ group.</p

FTS promotes DHA/ARS-induced caspase-8 and -9 activations.

<p>(<b>A</b> and <b>B</b>) <i>I</i>_CFP<i>/I</i>_YFP/Venus ratio images of cells expressing FRET-Bid or SCAT9 (left) and corresponding statistical results (right) from at least 250 cells in Huh-7 (<b>A</b>) and HepG2 (<b>B</b>) cells. Cells were transfected with FRET-Bid and SCAT9 plasmid for 24 h respectively and then treated with DHA/ARS for 24 h in the presence or absence of FTS before fluorescence microscopic imaging. *<i>P</i> < 0.05 and ***<i>P</i> < 0.001, compared with control; ^#<i>P</i> < 0.05, ^##<i>P</i> < 0.01 and ^###<i>P</i> < 0.001. (<b>C</b> and <b>D</b>) Cells were pretreated with Z-ITED-FMK (caspase-8 inhibitor, 20 μM) for 30 min before FTS/DHA/ARS treatment or the combination treatment of DHA/ARS and FTS for 48 h and then analyzed by CCK-8 assay. *<i>P</i> < 0.05, **<i>P</i> < 0.01, and ***<i>P</i> < 0.001.</p