Xiao Qi Wang

Topical delivery of siRNA-based spherical nucleic acid nanoparticle conjugates for gene regulation

Topical application of nucleic acids offers many potential therapeutic advantages for suppressing genes in the skin, and potentially for systemic gene delivery. However, the epidermal barrier typically precludes entry of gene-suppressing therapy unless the barrier is disrupted. We now show that spherical nucleic acid nanoparticle conjugates (SNA-NCs), gold cores surrounded by a dense shell of highly oriented, covalently immobilized siRNA, freely penetrate almost 100% of keratinocytes in vitro, mouse skin, and human epidermis within hours after application. Significantly, these structures can be delivered in a commercial moisturizer or phosphate-buffered saline, and do not require barrier disruption or transfection agents, such as liposomes, peptides, or viruses. SNA-NCs targeting epidermal growth factor receptor (EGFR), an important gene for epidermal homeostasis, are > 100-fold more potent and suppress longer than siRNA delivered with commercial lipid agents in cultured keratinocytes. Topical delivery of 1.5 uM EGFR siRNA (50 nM SNA-NCs) for 3 wk to hairless mouse skin almost completely abolishes EGFR expression, suppresses downstream ERK phosphorylation, and reduces epidermal thickness by almost 40%. Similarly, EGFR mRNA in human skin equivalents is reduced by 52% after 60 h of treatment with 25 nM EGFR SNA-NCs. Treated skin shows no clinical or histological evidence of toxicity. No cytokine activation in mouse blood or tissue samples is observed, and after 3 wk of topical skin treatment, the SNA structures are virtually undetectable in internal organs. SNA conjugates may be promising agents for personalized, topically delivered gene therapy of cutaneous tumors, skin inflammation, and dominant negative genetic skin disorders.

Soft, curved electrode systems capable of integration on the auricle as a persistent brain-computer interface

Significance Conventional electroencephalogram (EEG) recording systems, particularly the hardware components that form the physical interfaces to the head, have inherent drawbacks that limit the widespread use of continuous EEG measurements for medical diagnostics, sleep monitoring, and cognitive control. Here we introduce soft electronic constructs designed to intimately conform to the complex surface topology of the auricle and the mastoid, to provide long-term, high-fidelity recording of EEG data. Systematic studies reveal key aspects of the extreme levels of bending and stretching that are involved in mounting on these surfaces. Examples in persistent brain–computer interfaces, including text spellers with steady-state visually evoked potentials and event-related potentials, with viable operation over periods of weeks demonstrate important advances over alternative brain–computer interface technologies. Recent advances in electrodes for noninvasive recording of electroencephalograms expand opportunities collecting such data for diagnosis of neurological disorders and brain–computer interfaces. Existing technologies, however, cannot be used effectively in continuous, uninterrupted modes for more than a few days due to irritation and irreversible degradation in the electrical and mechanical properties of the skin interface. Here we introduce a soft, foldable collection of electrodes in open, fractal mesh geometries that can mount directly and chronically on the complex surface topology of the auricle and the mastoid, to provide high-fidelity and long-term capture of electroencephalograms in ways that avoid any significant thermal, electrical, or mechanical loading of the skin. Experimental and computational studies establish the fundamental aspects of the bending and stretching mechanics that enable this type of intimate integration on the highly irregular and textured surfaces of the auricle. Cell level tests and thermal imaging studies establish the biocompatibility and wearability of such systems, with examples of high-quality measurements over periods of 2 wk with devices that remain mounted throughout daily activities including vigorous exercise, swimming, sleeping, and bathing. Demonstrations include a text speller with a steady-state visually evoked potential-based brain–computer interface and elicitation of an event-related potential (P300 wave).

Suppression of Epidermal Growth Factor Receptor Signaling by Protein Kinase C-α Activation Requires CD82, Caveolin-1, and Ganglioside

Activation of protein kinase C (PKC)-alpha decreases normal and neoplastic cell proliferation by inhibiting epidermal growth factor receptor (EGFR)-related signaling. The molecular interactions upstream to PKC-alpha that influence its suppression of EGFR, however, are poorly understood. We have found that caveolin-1, tetraspanin CD82, and ganglioside GM3 enable the association of EGFR with PKC-alpha, ultimately leading to inhibition of EGFR signaling. GM3- and CD82-induced inhibition of EGFR signaling requires PKC-alpha translocation and serine/threonine phosphorylation, which eventually triggers EGFR Thr654 phosphorylation and receptor internalization. Within this ordered complex of signaling molecules, the ability of CD82 to associate with PKC-alpha requires the presence of caveolin-1, whereas the interaction of caveolin-1 or PKC-alpha with EGFR requires the presence of CD82 and ganglioside GM3. Disruption of the membrane with methyl-beta-cyclodextrin dissociates the EGFR/GM3/caveolin-1/CD82/PKC-alpha complex and prevents the inhibitory effect of PKC-alpha on EGFR phosphorylation, suggesting that caveolin-1, CD82, and ganglioside interact with EGFR and PKC-alpha within intact cholesterol-enriched membrane microdomains. Given the role of these membrane molecules in suppressing EGFR signaling, up-regulation of GM3, caveolin-1, and CD82 function may be an effective adjunctive therapy for treating epithelial cell malignancies.

siRNA-based spherical nucleic acids reverse impaired wound healing in diabetic mice by ganglioside GM3 synthase knockdown

Significance Diabetic patients often suffer from impaired wound healing, which can develop into nonhealing diabetic ulcers, facilitate bacterial infections, and necessitate amputation. Current strategies for treatment have failed to achieve the anticipated efficacy and do not address the fundamental molecular abnormalities that prevent efficient wound closure. In this work, we introduce a previously unidentified approach to treating diabetic wound healing by using topically delivered spherical nucleic acids to effect the knockdown of ganglioside-monosialic acid 3 (GM3) synthase, a mediator of impaired wound healing, in type 2 diabetic mice. In addition to laying the groundwork for developing a therapy for a debilitating condition, this work also validates the critical role of GM3 in diabetic wound healing. Spherical nucleic acid (SNA) gold nanoparticle conjugates (13-nm-diameter gold cores functionalized with densely packed and highly oriented nucleic acids) dispersed in Aquaphor have been shown to penetrate the epidermal barrier of both intact mouse and human skin, enter keratinocytes, and efficiently down-regulate gene targets. ganglioside-monosialic acid 3 synthase (GM3S) is a known target that is overexpressed in diabetic mice and responsible for causing insulin resistance and impeding wound healing. GM3S SNAs increase keratinocyte migration and proliferation as well as insulin and insulin-like growth factor-1 (IGF1) receptor activation under both normo- and hyperglycemic conditions. The topical application of GM3S SNAs (50 nM) to splinted 6-mm-diameter full-thickness wounds in diet-induced obese diabetic mice decreases local GM3S expression by >80% at the wound edge through an siRNA pathway and fully heals wounds clinically and histologically within 12 d, whereas control-treated wounds are only 50% closed. Granulation tissue area, vascularity, and IGF1 and EGF receptor phosphorylation are increased in GM3S SNA-treated wounds. These data capitalize on the unique ability of SNAs to naturally penetrate the skin and enter keratinocytes without the need for transfection agents. Moreover, the data further validate GM3 as a mediator of the delayed wound healing in type 2 diabetes and support regional GM3 depletion as a promising therapeutic direction.

Carbohydrate-Carbohydrate Binding of Ganglioside to Integrin α5 Modulates α5β1Function

Gangliosides GT1b and GD3, components of keratinocyte membranes, inhibit keratinocyte adhesion to fibronectin. Although ganglioside sialylation is known to be important, the mechanism of inhibition is unknown. Using purified insect recombinant α5 and β1 proteins and α5β1 integrin from lysed keratinocyte-derived SCC12 cells, we have shown that GT1b and GD3 inhibit the binding of α5β1 to fibronectin. Co-immunoprecipitation of GT1b and α5β1from SCC12 cells and direct binding of GT1b and GD3 to affinity-purified α5β1 from SCC12 cells and insect recombinant α5β1, particularly the α5 subunit, further suggest interaction between ganglioside and α5β1. The carbohydrate moieties of integrin appear to be critical since gangliosides are unable to bind deglycosylated forms of α5β1from SCC12 and insect cells or poorly glycosylated recombinant α5β1 from Escherichia colicells. The GT1b-α5β1 interaction is inhibited by concanavalin A, suggesting that GT1b binds to mannose structures in α5β1. The preferential binding of GT1b to high mannose rather than reduced mannose ovalbumin further implicates the binding of GT1b to mannose structures. These data provide evidence that highly sialylated gangliosides regulate α5β1-mediated adhesion of epithelial cells to fibronectin through carbohydrate-carbohydrate interactions between GT1b and the α5 subunit of α5β1 integrin.

Ginsenoside Rg3 inhibits epithelial-mesenchymal transition (EMT) and invasion of lung cancer by down-regulating FUT4

The epithelial-mesenchymal transition (EMT) is an important factor in lung cancer metastasis, and targeting EMT is a potential therapeutic strategy. Fucosyltransferase IV (FUT4) and its synthetic cancer sugar antigen Lewis Y (LeY) was abnormally elevated in many cancers. In this study, a traditional Chinese medicine ginsenoside Rg3 was used to investigate whether its inhibition to EMT and invasion of lung cancer is by the glycobiology mechanism. We found that Rg3 treatment (25, 50, 100 μg/ml) inhibited cell migration and invasion by wound-healing and transwell assays. Rg3 could significantly alter EMT marker proteins with increased E-cadherin, but decreased Snail, N-cadherin and Vimentin expression. Rg3 also down-regulated FUT4 gene and protein expression in lung cancer cells by qPCR, Western blot and immunofluorescence. After FUT4 down-regulated with shFUT4, EMT was obviously inhibited. Furthermore, the activation of EGFR through decreased LeY biosynthesis was inhibited, which blocked the downstream MAPK and NF-κB signal pathways. In addition, Rg3 reduced tumor volume and weight in xenograft mouse model, and significantly decreased tumor metastasis nodules in lung tissues by tail vein injection. In conclusion, Rg3 inhibits EMT and invasion of lung cancer by down-regulating FUT4 mediated EGFR inactivation and blocking MAPK and NF-κB signal pathways. Rg3 may be a potentially effective agent for the treatment of lung cancer.

Ganglioside Modulation Regulates Epithelial Cell Adhesion and Spreading via Ganglioside-specific Effects on Signaling

Gangliosides are implicated in regulating cell adhesion and migration on fibronectin by binding with the α5 subunit of α5β1integrin. However, the effects of gangliosides on cell spreading and related signaling pathways are unknown. Increases in gangliosides GT1b and GD3 inhibited spreading on fibronectin, concurrent with inhibition of Src and focal adhesion kinase. Although antibody blockade of GT1b or GD3 function and gene-modulated ganglioside depletion stimulated spreading and activated Src and focal adhesion kinase, the augmented spreading by disruption of GT1b function, but not by disruption of GD3 function, was inhibited by blockade of Src and focal adhesion kinase activation. In contrast, inhibitors of protein kinase C prevented the stimulation of spreading by GD3 functional inhibition, but not by GT1b functional blockade. Modulation of either GT1b or GD3 content affected phosphoinositol 3-kinase activation, and inhibition of this activation reversed the stimulation of cell spreading by anti-GD3 antibody, anti-GT1b antibody, and ganglioside depletion, suggesting that phosphoinositol 3-kinase is an intermediate in both the FAK/Src and protein kinase C pathways that lead to cell spreading. These studies demonstrate that epithelial cell ganglioside GT1b modulates cell spreading through α5β1/FAK and phosphoinositol 3-kinase signaling, whereas GD3-modulated spreading appears to involve phosphoinositol 3-kinase-dependent protein kinase C signaling.

Ginsenoside Rg3 Inhibits Melanoma Cell Proliferation through Down-Regulation of Histone Deacetylase 3 (HDAC3) and Increase of p53 Acetylation

Malignant melanoma is an aggressive and deadly form of skin cancer, and despite recent advances in available therapies, is still lacking in completely effective treatments. Rg3, a monomer extracted from ginseng roots, has been attempted for the treatment of many cancers. It is reported that the expressions of histone deacetylase 3 (HDAC3) and p53 acetylation correlate with tumor cell growth. However, the antitumor effect of Rg3 on melanoma and the mechanism by which it regulates HDAC3 expression and p53 acetylation remain unknown. We found high expression of HDAC3 in human melanoma tissues to be significantly correlated to lymph node metastasis and clinical stage of disease (p<0.05). In melanoma cells, Rg3 inhibited cell proliferation and induced G0/G1 cell cycle arrest. Rg3 also decreased the expression of HDAC3 and increased the acetylation of p53 on lysine (k373/k382). Moreover, suppression of HDAC3 by either siRNA or a potent HDAC3 inhibitor (MS-275) inhibited cell proliferation, increased p53 acetylation and transcription activity. In A375 melanoma xenograft studies, we demonstrated that Rg3 and HDAC3 short hairpin RNA (shHDAC3) inhibited the growth of xenograft tumors with down-regulation of HDAC3 expression and up-regulation of p53 acetylation. In conclusion, Rg3 has antiproliferative activity against melanoma by decreasing HDAC3 and increasing acetylation of p53 both in vitro and in vivo. Thus, Rg3 serves as a potential therapeutic agent for the treatment of melanoma.

Overexpression of fucosyltransferase IV promotes A431 cell proliferation through activating MAPK and PI3K/Akt signaling pathways

Lewis Y (LeY) is a carbohydrate tumor‐asssociated antigen. The majority of cancer cells derived from epithelial tissue express LeY type difucosylated oligosaccharide. Fucosyltransferase IV (FUT4) is an essential enzyme that catalyzes the synthesis of LeY oligosaccharide. Our previous studies have shown that FUT4 overexpression promotes A431 cell proliferation, but the mechanism is still largely unknown. Herein, we investigated the role of the mitogen‐activated protein kinases (MAPKs) and phosphoinositide‐3 kinase (PI3K)/Akt signaling pathways on FUT4‐induced cell proliferation. Results show that overexpression of FUT4 increases the phosphorylation of ERK1/2, p38 MAPK, and PI3K/Akt. Inhibitors of PI3K (LY294002 and Wortmannin) prevented the phosphorylation of ERK1/2, p38 MAPK, and Akt PI3K). Moreover, phosphorylation of Akt is abolished by inhibitors of ERK1/2 (PD98059) and p38 MAPK (SB203580). These data suggested that FUT4 not only activates MAPK and PI3K/Akt signals, but also promotes the crosstalk among these signaling pathways. In addition, FUT4‐induced stimulation of cell proliferation correlates with increased cell cycle progression by promoting cells into S‐phase. The mechanism involves in increased expression of cyclin D1, cyclin E, CDK 2, CDK 4, and pRb, and decreased level of cyclin‐dependent kinases inhibitors p21 and p27, which are blocked by the inhibitors of upstream signal molecules, MAPK and PI3K/Akt. In conclusion, these studies suggest that FUT4 regulates A431 cell growth through controlling cell cycle progression via MAPK and PI3K/Akt signaling pathways. J. Cell. Physiol. 225: 612–619, 2010.

De-N-acetyl GM3 Promotes Melanoma Cell Migration and Invasion through Urokinase Plasminogen Activator Receptor Signaling–Dependent MMP-2 Activation

We have recently discovered that de-N-acetyl GM3 [NeuNH(2)LacCer, d-GM3], a derivative of ganglioside GM3, is specifically expressed in metastatic tumor cells and that its expression correlates with an enhanced metastatic phenotype. Although the classic N-acetylated form of GM3 (NeuAcLacCer, c-GM3) is found in both normal and tumor cells, metastatic tumor cells (but not other cells) predominantly express d-GM3 (82-95% of total GM3). d-GM3 expression is mainly found in metastatic melanomas, but not in benign nevi or the majority of primary melanomas. Using metastatic (d-GM3-positive) and poorly invasive (d-GM3-negative) human melanoma cell lines, we found that d-GM3 stimulates cell migration and invasion by increasing the expression and activation of urokinase-like plasminogen activator (uPA). Further studies showed that d-GM3 activates matrix metalloproteinase-2 (MMP-2), but not MMP-9, when uPA receptor signaling is activated. These results implicate d-GM3 as a specific marker for metastatic melanoma and a novel therapeutic target for neoplastic diseases.