Wenjie Guo

Target‐Cell‐Specific Delivery, Imaging, and Detection of Intracellular MicroRNA with a Multifunctional SnO2 Nanoprobe

MicroRNAs (miRNAs) are a class of short, endogenous, noncoding regulatory RNAs (approximately 18–25 nucleotides), encoded in the genomes of plants, animals, and viruses. They partially complement the 3’ untranslated region of target mRNAs, causing mRNA cleavage or inhibiting protein synthesis within the Dicer/Argonaute complex. MiRNAs play key regulatory roles in a diverse range of biological processes, including cell development, differentiation, metabolism, and apoptosis. In particular, distinct miRNA expression patterns are associated with various cancer phenotypes. Therefore, miRNAs are also an emerging class of useful diagnostic and prognostic markers. However, miRNA analysis is challenging owing to the unique characteristics of miRNAs, such as their small size, sequence similarity among family members, low abundance, susceptibility to degradation, and the technical impediments in delivering miRNAs across the plasma membrane of cells. An improved delivery strategy is thus needed to successfully monitor intracellular miRNA levels in situ. Generally, transmission of oligonucleotides for gene therapy falls into two broad categories, viral vectors or nonviral carriers. Viral vectors exhibit high transfection efficiency but also some fundamental problems, such as immunogenicity and toxicity. These problems limit their broad application. Currently, the nonviral vectors, such as liposomes, cationic polymers, dendrimers, polypeptides, and nanomaterials have attracted significant interest, owing to their good biocompatibility and potential for large-scale production, in contrast to viral vectors. However, many of these carrier systems do not inhibit genes in a cell-specific manner and cannot be used to monitor their intracellular delivery or therapeutic response. An excellent nonviral vector should efficiently facilitate cell-specific gene-probe (a single-stranded oligonucleotide designed to recognize a single target-nucleotide sequence) uptake and gene-probe endosomal escape for intracellular delivery. Fluorescent nanoparticles are highly attractive materials for gene-probe delivery because of their unique properties, including uniform size, superior imaging characteristics, and facile surface modification. The rational functionalization of these nanomaterials with target-specific moieties, such as antibodies, aptamers, and other molecules, to recognize receptors on the cell surface has led to versatile theragnostic nanosystems. These multifunctional nanosystems not only allow efficient delivery of gene probes to target cells with fewer side effects, but also allow the simultaneous monitoring of delivery and the therapeutic response of the targeted genes, using the advanced optical properties of the nanosystem. However, these nanosystems have not been used for in situ detection of intracellular miRNAs yet, because the detection strategy has not been optimized. In this study we design a novel multifunctional SnO2 nanoprobe (mf-SnO2), which contains a cell-targeting moity, as well as a conjugated gene probe to specifically recognize the target sequence, thus providing a detection strategy or inhibitor. Also, visualization of the delivery and intracellular response is possible through fluorescence of the SnO2. As shown in Scheme 1, cell-specific delivery is achieved by functionalizing SnO2 nanoparticles (SnO2NPs) with folic acid (FA), which targets cancer cells; a gene probe, in this case a molecular beacon (MB) to detect target miRNAs, is conjugated by a disulfide linkage, which is sensitive to pH values. Cleavage of the disulfide linkage between the gene probe and the nanoparticle enhances the efficiency of intracellular delivery. Using miRNA-21 in HeLa cells as a model, a method for in situ detection of intracellular miRNA by the multifunctional nanoprobe is reported. To verify the practicality of this approach, another nanoprobe was also designed, by substituting the MB with an anti-miR of miRNA-21, to down-regulate the expression of a target miRNA. The proposed method, with a MB as the recognition probe, can be used subsequently to monitor the change in miRNA levels from negligible cytotoxicity, and to monitor the ability of the multifunctional nanoprobe to [*] Dr. H. Dong, Prof. J. Lei, Prof. H. Ju, Z. Zhu State Key Laboratory of Analytical Chemistry for Life Science, School of Chemistry and Chemical Engineering, Nanjing University Nanjing 210093 (P.R. China) E-mail: hxju@nju.edu.cn

Functionalized Graphene Oxide Mediated Adriamycin Delivery and miR-21 Gene Silencing to Overcome Tumor Multidrug Resistance In Vitro

Multidrug resistance (MDR) is a major impediment to successful cancer chemotherapy. Co-delivery of novel MDR-reversing agents and anticancer drugs to cancer cells holds great promise for cancer treatment. MicroRNA-21 (miR-21) overexpression is associated with the development and progression of MDR in breast cancer, and it is emerging as a novel and promising MDR-reversing target. In this study, a multifunctional nanocomplex, composed of polyethylenimine (PEI)/poly(sodium 4-styrenesulfonates) (PSS)/graphene oxide (GO) and termed PPG, was prepared using the layer-by-layer assembly method to evaluate the reversal effects of PPG as a carrier for adriamycin (ADR) along with miR-21 targeted siRNA (anti-miR-21) in cancer drug resistance. ADR was firstly loaded onto the PPG surface (PPGADR) by physical mixing and anti-miR-21 was sequentially loaded onto PPGADR through electric absorption to form anti-miR-21PPGADR. Cell experiments showed that PPG significantly enhanced the accumulation of ADR in MCF-7/ADR cells (an ADR resistant breast cancer cell line) and exhibited much higher cytotoxicity than free ADR, suggesting that PPG could effectively reverse ADR resistance of MCF-7/ADR. Furthermore, the enhanced therapeutic efficacy of PPG could be correlated with effective silencing of miR-21 and with increased accumulation of ADR in drug-resistant tumor cells. The endocytosis study confirmed that PPG could effectively carry drug molecules into cells via the caveolae and clathrin-mediated endocytosis pathways. These results suggest that this PPG could be a potential and efficient non-viral vector for reversing MDR, and the strategy of combining anticancer drugs with miRNA therapy to overcome MDR could be an attractive approach in cancer treatment.

Novel monofunctional platinum (II) complex Mono-Pt induces apoptosis-independent autophagic cell death in human ovarian carcinoma cells, distinct from cisplatin

Failure to engage apoptosis appears to be a leading mechanism of resistance to traditional platinum drugs in patients with ovarian cancer. Therefore, an alternative strategy to induce cell death is needed for the chemotherapy of this apoptosis-resistant cancer. Here we report that autophagic cell death, distinct from cisplatin-induced apoptosis, is triggered by a novel monofunctional platinum (II) complex named Mono-Pt in human ovarian carcinoma cells. Mono-Pt-induced cell death has the following features: cytoplasmic vacuolation, caspase-independent, no nuclear fragmentation or chromatin condensation, and no apoptotic bodies. These characteristics integrally indicated that Mono-Pt, rather than cisplatin, initiated a nonapoptotic cell death in Caov-3 ovarian carcinoma cells. Furthermore, incubation of the cells with Mono-Pt but not with cisplatin produced an increasing punctate distribution of microtubule-associated protein 1 light chain 3 (LC3), and an increasing ratio of LC3-II to LC3-I. Mono-Pt also caused the formation of autophagic vacuoles as revealed by monodansylcadaverine staining and transmission electron microscopy. In addition, Mono-Pt-induced cell death was significantly inhibited by the knockdown of either BECN1 or ATG7 gene expression, or by autophagy inhibitors 3-methyladenine, chloroquine and bafilomycin A1. Moreover, the effect of Mono-Pt involved the AKT1-MTOR-RPS6KB1 pathway and MAPK1 (ERK2)/MAPK3 (ERK1) signaling, since the MTOR inhibitor rapamycin increased, while the MAPK1/3 inhibitor U0126 decreased Mono-Pt-induced autophagic cell death. Taken together, our results suggest that Mono-Pt exerts anticancer effect via autophagic cell death in apoptosis-resistant ovarian cancer. These findings lead to increased options for anticancer platinum drugs to induce cell death in cancer.

Inhibition of Th1/Th17 responses via suppression of STAT1 and STAT3 activation contributes to the amelioration of murine experimental colitis by a natural flavonoid glucoside icariin

Inflammatory bowel disease (IBD) is a chronic inflammatory disorder in the intestine which involves overproduction of pro-inflammatory cytokines and excessive functions of inflammatory cells. However, current treatments for IBD may have potential adverse effects including steroid dependence, infections and lymphoma. Therefore new therapies or drug candidates for the treatment of IBD are desperately needed. In the present study we found that icariin, a major bioactive compound from plants in Epimedium family, exerted protective effect on intestinal inflammation in mice induced by dextran sulfate sodium. Oral administration of icariin significantly attenuated the disease progression and alleviated the pathological changes of colitis. It also inhibited the production of pro-inflammatory cytokines and expression of p-p65, p-STAT1 and p-STAT3 in colon tissues. Further study showed that icariin dose-dependently inhibited the proliferation and activation of T lymphocytes, and suppressed pro-inflammatory cytokine levels of activated T cells. Moreover, icariin treatment inhibited the phosphorylations of STAT1 and STAT3 in CD4(+) T cells, which were the crucial transcription factors for Th1 and Th17 respectively. Taken together, these results indicate that icariin is a potential therapeutic agent for IBD.

A novel benzo[d]imidazole derivate prevents the development of dextran sulfate sodium-induced murine experimental colitis via inhibition of NLRP3 inflammasome

NLRP3 inflammasome has been reported to be associated with various kinds of immunological diseases including colitis. However, there are few drug candidates targeting inflammasomes for the treatment of colitis. In the present study, we aimed at examining the effect of 1-ethyl-5-methyl-2-phenyl-1H-benzo[d]imidazole, a synthetic small molecular compound also named Fc11a-2, for the treatment of dextran sulfate sodium (DSS)-induced experimental colitis in mice via targeting NLRP3 inflammasome. Treatment with Fc11a-2 dose-dependently attenuated the loss of body weight and shortening of colon length induced by DSS. In addition, the disease activity index, histopathologic scores and myeloperoxidase activity were also significantly reduced by Fc11a-2 treatment. Moreover, protein and mRNA levels of DSS-induced proinflammatory cytokines in colon, including TNF-α, IL-1β, IL-18, IL-17A and IFN-γ, were markedly suppressed by Fc11a-2. Furthermore, a decreased CD11c⁺ macrophage infiltration in colons and inactivation of caspase-1 in peritoneal macrophages were detected in Fc11a-2-treated mice. The mechanism of action of Fc11a-2 was related to the inhibition of the cleavage of pro-caspase-1, pro-IL-1β and pro-IL-18 which in turn suppressed the activation of NLRP3 inflammasome. Taken together, our results demonstrate the ability of Fc11a-2 to inhibit NLRP3 inflammasome activation and its potential use in the treatment of inflammatory bowel diseases.

Selective Sequestration of STAT1 in the Cytoplasm via Phosphorylated SHP-2 Ameliorates Murine Experimental Colitis

The side effects of current immunosuppressive drugs have impeded the development of therapies for immune diseases. Selective regulation of STAT signaling is an attractive strategy for treating immune disorders. In this study, we used a small-molecule compound to explore possible means of targeting STAT1 for the treatment of Th1-mediated inflammation. Selective regulation of STAT1 signaling in T cells from C57BL/6 mice was accomplished using fusaruside, a small-molecule compound that triggers the tyrosine phosphorylation of Src homology 2-containing protein tyrosine phosphatase 2 (SHP-2). The interaction of tyrosine phosphorylated SHP-2 (pY-SHP-2) with cytosolic STAT1 prevented the recruitment of STAT1 to IFN-γR and specifically inhibited STAT1 signaling, resulting in a reduction in Th1 cytokine production and an improvement in 2, 4, 6-trinitrobenzene sulfonic acid-induced colitis in mice. Blocking the pY-SHP-2–STAT1 interaction, with SHP-2 inhibitor NSC-87877 or using T cells from conditional SHP-2 knockout mice, reversed the effects of fusaruside, resulting in STAT1 activation and worsened colitis. The fusaruside-induced ability of pY-SHP-2 to selectively sequestrate STAT1 from recruitment to the receptor is independent of its function as a phosphatase, demonstrating a novel role for SHP-2 in regulating both STAT1 signaling and Th1-type immune responses. These findings could lead to increased options for the treatment of Crohn’s disease and other Th1-mediated inflammatory diseases.

Suppression of NF-κB signaling and NLRP3 inflammasome activation in macrophages is responsible for the amelioration of experimental murine colitis by the natural compound fraxinellone

Inflammatory bowel disease (IBD) affects millions of people worldwide. Although the etiology of this disease is uncertain, accumulating evidence indicates a key role for the activated mucosal immune system. In the present study, we examined the effects of the natural compound fraxinellone on dextran sulfate sodium (DSS)-induced colitis in mice, an animal model that mimics IBD. Treatment with fraxinellone significantly reduced weight loss and diarrhea in mice and alleviated the macroscopic and microscopic signs of the disease. In addition, the activities of myeloperoxidase and alkaline phosphatase were markedly suppressed, while the levels of glutathione were increased in colitis tissues following fraxinellone treatment. This compound also decreased the colonic levels of interleukin (IL)-1β, IL-6, IL-18 and tumor necrosis factor (TNF)-α in a concentration-dependent manner. These effects of fraxinellone in mice with experimental colitis were attributed to its inhibition of CD11b(+) macrophage infiltration. The mRNA levels of macrophage-related molecules in the colon, including intercellular adhesion molecule 1 (ICAM1), vascular cell adhesion molecule 1 (VCAM1), inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX2), were also markedly inhibited following fraxinellone treatment. The results from in vitro assays showed that fraxinellone significantly reduced lipopolysaccharide (LPS)-induced production of nitric oxide (NO), IL-1β and IL-18 as well as the activity of iNOS in both THP-1 cells and mouse primary peritoneal macrophages. The mechanisms responsible for these effects were attributed to the inhibitory role of fraxinellone in NF-κB signaling and NLRP3 inflammasome activation. Overall, our results support fraxinellone as a novel drug candidate in the treatment of colonic inflammation.

Andrographolide sulfonate ameliorates experimental colitis in mice by inhibiting Th1/Th17 response

Inflammatory bowel disease (IBD) is a chronic, relapsing and remitting condition of inflammation involves overproduction of pro-inflammatory cytokines and excessive functions of inflammatory cells. However, current treatments for IBD may have potential adverse effects including steroid dependence, infections and lymphoma. Therefore new therapies for the treatment of IBD are desperately needed. In the present study, we aimed to examine the effect of andrographolide sulfonate, a water-soluble form of andrographolide (trade name: Xi-Yan-Ping Injection), on murine experimental colitis induced by 2, 4, 6-trinitrobenzene sulfonic acid (TNBS). Andrographolide sulfonate was administrated through intraperitoneal injection to mice with TNBS-induced colitis. TNBS-induced body weight loss, myeloperoxidase activity, shortening of the colon and colonic inflammation were significantly ameliorated by andrographolide sulfonate. Both the mRNA and protein levels of pro-inflammatory cytokines were reduced by andrographolide sulfonate administration. Moreover, andrographolide sulfonate markedly suppressed the activation of p38 mitogen-activated protein kinase as well as p65 subunit of nuclear factor-κB (NF-κB). Furthermore, CD4(+) T cell infiltration as well as the differentiation of Th1 (CD4(+)IFN-γ(+)) and Th17 (CD4(+)IL17A(+)) subset were inhibited by andrographolide sulfonate. In summary, these results suggest that andrographolide sulfonate ameliorated TNBS-induced colitis in mice through inhibiting Th1/Th17 response. Our study shows that water-soluble andrographolide sulfonate may represent a new therapeutic approach for treating gastrointestinal inflammatory disorders.

Water-soluble andrographolide sulfonate exerts anti-sepsis action in mice through down-regulating p38 MAPK, STAT3 and NF-κB pathways

Andrographolide is a prescribed drug used for preventing and treating the common cold, influenza, viral infections or allergies. However, its poor water solubility enormously limits its bioavailability. In the present study, we aimed at examining and comparing the effect of andrographolide sulfonate (trade name: Xi-Yan-Ping Injection), a water-soluble form made from andrographolide through sulfonating reaction, on the treatment of murine sepsis model induced by lipopolysaccharide (LPS). Pretreatment with andrographolide sulfonate significantly decreased the levels of tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β) and transaminase activities in serum, attenuated liver and lung damage, and improved the survival of mice with experimental sepsis. Andrographolide sulfonate also remarkably reduced the expression levels of TNF-α, IL-1β, IL-6 and inducible nitric oxide synthase in the injured liver from septic mice. Moreover, andrographolide sulfonate time-dependently suppressed the activation of p38 mitogen-activated protein kinase (MAPK) but not extracellular signal-regulated kinase (ERK1/2) or c-Jun NH(2)-terminal kinase (JNK). Furthermore, pretreatment with andrographolide sulfonate markedly inhibited the activation of p65 subunit of nuclear factor-κB (NF-κB) as well as signal transducers and activators of transcription 3 (STAT3) in the injured liver from mice with endotoxic shock. Notably, andrographolide sulfonate showed a much stronger alleviation of LPS-induced sepsis in mice compared with andrographolide. Taken together, these results reveal that andrographolide sulfonate ameliorates sepsis in mice through suppressing p38 MAPK, STAT3 and NF-κB pathways and suggest that andrographolide sulfonate has an advantage of andrographolide for the treatment of endotoxin shock.

A Novel Chromone Derivative with Anti-Inflammatory Property via Inhibition of ROS-Dependent Activation of TRAF6-ASK1-p38 Pathway

The p38 MAPK signaling pathway plays a pivotal role in inflammation. Targeting p38 MAPK may be a potential strategy for the treatment of inflammatory diseases. In the present study, we show that a novel chromone derivative, DCO-6, significantly reduced lipopolysaccharide (LPS)-induced production of nitric oxide, IL-1β and IL-6, decreased the levels of iNOS, IL-1β and IL-6 mRNA expression in both RAW264.7 cells and mouse primary peritoneal macrophages, and inhibited LPS-induced activation of p38 MAPK but not of JNK, ERK. Moreover, DCO-6 specifically inhibited TLR4-dependent p38 activation without directly inhibiting its kinase activity. LPS-induced production of intracellular reactive oxygen species (ROS) was remarkably impaired by DCO-6, which disrupted the formation of the TRAF6-ASK1 complex. Administering DCO-6 significantly protected mice from LPS-induced septic shock in parallel with the inhibition of p38 activation and ROS production. Our results indicate that DCO-6 showed anti-inflammatory properties through inhibition of ROS-dependent activation of TRAF6-ASK1-p38 pathway. Blockade of the upstream events required for p38 MAPK action by DCO-6 may provide a new therapeutic option in the treatment of inflammatory diseases.