Xinglei Yao

The effect of surface modification of amorphous silica particles on NLRP3 inflammasome mediated IL-1β production, ROS production and endosomal rupture

Although amorphous silica particles (SPs) are widely used in cosmetics, foods and medicinal products, it has gradually become evident that SPs can induce substantial inflammation accompanied by interleukin-1beta (IL-1beta) production. Here, to develop safe forms of SPs, we examined the mechanisms of SP-induced inflammation and the relationship between particle characteristics and biological responses. We compared IL-1beta production levels in THP-1 human macrophage like cells in response to unmodified SP of various diameters (30- to 1000-nm) and demonstrated that unmodified microsized 1000-nm SP (mSP1000) induced higher levels of IL-1beta production than did smaller unmodified SPs. Furthermore, we found that unmodified mSP1000-induced IL-1beta production was depended on the sequence of reactive oxygen species (ROS) production, endosomal rupture, and subsequent activation of pro-inflammatory complex NLRP3 inflammasome. In addition, we compared IL-1beta production levels in THP-1 cells treated with mSP1000s modified with a functional group (-COOH, -NH(2), -SO(3)H, -CHO). Although unmodified and surface-modified mSP1000s were taken up with similar frequencies equally into the THP-1 cells, surface modification of mSP1000 dramatically suppressed IL-1beta production by reducing ROS production. Our results reveal a part of NLRP3 activation pathway and provide basic information that should help to create safe and effective forms of SPs.

Titanium dioxide induces different levels of IL-1β production dependent on its particle characteristics through caspase-1 activation mediated by reactive oxygen species and cathepsin B

Although titanium dioxide (TiO2) is widely used, its inhalation can induce inflammatory diseases accompanied by interleukin-1beta (IL-1beta) production. The particle characteristics of TiO2 are important factors in its biological effects. It is urgently necessary to investigate the relationship between the particle characteristics and biological responses for the development of safe forms of TiO2. Here, we systematically compared the production of IL-1beta in response to various forms of TiO2 by macrophage-like human THP-1 cells using various sizes (nano to micro), crystal structures (anatase or rutile), and shapes (spherical or spicular) of TiO2. The production of IL-1beta depended dramatically on the characteristics of the TiO2. Notably, smaller anatase and larger rutile particles provoked higher IL-1beta production. In addition, IL-1beta production depended on active cathepsin B and reactive oxygen species production independent of the characteristics of TiO2. Our results provide basic information for the creation of safe and effective novel forms of TiO2.

Systemic administration of a PEGylated adenovirus vector with a cancer-specific promoter is effective in a mouse model of metastasis

Cancer gene therapy by adenovirus vectors (Advs) for metastatic cancer is limited because systemic administration of Adv produces low therapeutic effect and severe side effects. In this study, we generated a dual cancer-specific targeting vector system by using PEGylation and the telomere reverse transcriptase (TERT) promoter and attempted to treat experimental metastases through systemic administration of the vectors. We first optimized the molecular size of PEG and modification ratios used to create PEG-Ads. Systemic administration of PEG-Ad with 20-kDa PEG at a 45% modification ratio (PEG[20K/45%]-Ad) resulted in higher tumor-selective transgene expression than unmodified Adv. Next, we examined the effectiveness against metastases and side effects of a TERT promoter-driven PEG[20K/45%]-Ad containing the herpes simplex virus thymidine kinase (HSVtk) gene (PEG-Ad-TERT/HSVtk). Systemic administration of PEG-Ad-TERT/HSVtk showed superior antitumor effects against metastases with negligible side effects. A cytomegalovirus (CMV) promoter-driven PEG[20K/45%]-Ad also produced antimetastatic effects, but these were accompanied by side effects. Combining PEG-Ad-TERT/HSVtk with etoposide or 5-fluorouracil enhanced the therapeutic effects with negligible side effects. These results suggest that modification with 20-kDa PEG at a 45% modification ratio is the optimal condition for PEGylation of Adv, and PEG-Ad-TERT/HSVtk is a prototype Adv for systemic cancer gene therapy against metastases.

Current Targeting Strategies for Adenovirus Vectors in Cancer Gene Therapy

Adenovirus vectors (Adv) are the most frequently used vectors in gene therapy research, especially in cancer gene therapy. However, despite encouraging preclinical and early clinical results, the successful clinical utility of gene therapy has not yet been fully realized. Challenges to clinical trial success for targeted Adv include inefficient Adv-mediated gene transfer (because many tumor cells lack Adv receptors), poor transduction in tumor tissues after systemic administration, accumulation and undesirable transgene expression in the liver. This review summarizes current targeting strategies for Adv to overcome these obstacles. Strategies include transductional selectivity through genetic modification of viral coat proteins, transcriptional selectivity by means of tumor-specific promoters, and selective biodistribution from conjugation with targeting ligands or polymers such as polyethylene glycol (PEG). Furthermore, combining selective biodistribution and active targeting ligands such as proteins, antibodies and peptides is an intriguing and promising approach that will also be covered in this review. These studies have provided new insights into our understanding of the utility of Adv in cancer gene therapy.

Tat conjugation of adenovirus vector broadens tropism and enhances transduction efficiency.

AIMS Adenovirus vectors (Advs) have been very useful for basic research and clinical gene therapy because they propagate to high titers and efficiently transduce cells and tissues regardless of the mitotic status. However, poor transduction of cells that lack the coxsackievirus and adenovirus receptor (CAR), the primary receptor for Advs, has limited Adv application. In this study, we attempted to generate novel Tat-Advs (Advs conjugated with the HIV Tat-derived peptide, a protein-transduction domain (PTD)) to broaden Adv tropism and enhance transduction efficiency. MAIN METHODS We constructed Tat-Advs by chemically conjugating Tat peptide to the surface-exposed lysine residues on Advs. We compared the gene transfer activity of Tat-Advs with that of unmodified Advs by measuring the luciferase expression in several types of cell lines. KEY FINDINGS Tat-Advs showed gene expression 1 to 3 log orders higher than unmodified Advs in CAR-negative adherent cells and blood cells, which are refractory to conventional Advs. The inhibition of Tat-Adv-mediated gene expression by heparin and macropinocytosis inhibitor confirms that binding of Tat-Adv to cellular HSPGs and macropinocytosis are essential for efficient CAR-independent transduction. We also demonstrated that Adv modified with another PTD (R8) had the same high transduction efficiency as Tat-Adv. SIGNIFICANCE These data suggest that Tat-Advs are important tools for transducing cells and will be useful as platform vectors for gene therapy.

Creation of a LIGHT mutant with the capacity to evade the decoy receptor for cancer therapy

The cytokine LIGHT activates various anti-tumor functions through its two receptors, lymphotoxin beta receptor (LTbetaR) and herpes virus entry mediator (HVEM), and is expected to be a promising candidate for cancer therapy. However, LIGHT is also trapped by decoy receptor 3 (DcR3), which is highly expressed in various tumors. Here, we used phage display technique to create LIGHT mutants that specifically bind LTbetaR and HVEM, and is not trapped by DcR3 for optimized cancer therapy. We constructed phage library displaying structural variants of LIGHT with randomized amino acid residues. After the affinity panning, we created 6 clones of LIGHT mutants as candidates for DcR3-evading LIGHT. Analysis of binding affinities showed that all candidates had 10-fold lower affinities for DcR3 than wild-type LIGHT, while 5 of the 6 clones had almost the same affinity for LTbetaR and HVEM. Furthermore, analysis of detailed binding kinetics showed that lower affinity for DcR3 is dependent on their faster off-rate. Further, we showed that the LIGHT mutant had almost the same cytotoxicity via LTbetaR, and had 62-fold higher DcR3-evading capacity compared to the wild type. Our data provide valuable information for construction of more functional LIGHT mutants that might be powerful tools for cancer therapy.

Creation of lysine-deficient mutant lymphotoxin-α with receptor selectivity by using a phage display system

The cytokine lymphotoxin-alpha (LT alpha) activates various biological functions through its three receptor subtypes, tumor necrosis factor receptor 1 (TNFR1), TNFR2 and herpes virus entry mediator (HVEM), but the relative contribution of each receptor to each function is unclear. Therefore it is important to create mutant LT alpha with receptor selectivity for optimized cancer therapy and the analysis of receptor function. Here, we attempted to create a lysine-deficient mutant LT alpha with TNFR1-selective bioactivity using a phage display technique. We obtained the TNFR1-selective mutant LT alpha R1selLT, which contained the mutations K19N, K28Q, K39S, K84Q, K89V, and K119H. Compared with wild-type LT alpha (wtLT alpha), R1selLT showed several-fold higher bioactivity via TNFR1 but 40-fold lower bioactivity via TNFR2. Kinetic association-dissociation parameters of R1selLT with TNFR2 were higher than those of wtLT alpha, whereas these parameters of R1selLT with TNFR1 were lower than those of wtLT alpha, suggesting that destabilization of the R1selLT-TNFR2 complex causes the decreased bioactivity of R1selLT on TNFR2. We also showed that the K84Q mutation contributed to the enhanced activity via TNFR1, and K39S lowered activity via TNFR2. R1selLT likely will be useful in cancer therapy and in analysis of the LT alpha structure-function relationship.

LIGHT protein suppresses tumor growth by augmentation of immune response

The tumor necrosis factor (TNF) superfamily member LIGHT has potent anti-tumor activities through activation of the immune response, and it is a promising candidate for use in cancer immunotherapy. However, there are no reports of the anti-tumor effects of LIGHT protein in vivo because of the lack of easy, efficient methods of manufacturing this protein. Here, we developed a method of manufacturing recombinant LIGHT protein using Escherichia coli through refolding of inclusion bodies; we then evaluated the anti-tumor activity of the protein. LIGHT protein expressed in E. coli showed the same biological activities and binding affinities to its receptors as did LIGHT expressed in mammalian cells. In addition, intratumoral injection of LIGHT significantly suppressed tumor growth, with augmentation of antigen-specific IFN-gamma-producing cells in the regional lymph nodes and spleen. These results indicate that LIGHT protein efficiently evokes the systemic tumor-specific immune response, and thus induces tumor suppression.

Creation of a lysine-deficient LIGHT mutant with the capacity for site-specific PEGylation and low affinity for a decoy receptor.

The cytokine LIGHT is a promising candidate for cancer therapy. However, the therapeutic effect of LIGHT as a systemic anticancer agent is currently insufficient because of its instability and its binding to nonfunctional soluble decoy receptor 3 (DcR3), which is overexpressed in various tumors. Modification of proteins with polyethylene glycol (PEGylation) can improve their in vivo stability, but PEGylation may occur randomly at all lysine residues and the NH(2)-terminus; therefore, PEGylated proteins are generally heterogeneous and have decreased bioactivity. In this study, we attempted to create a lysine-deficient LIGHT mutant that could be PEGylated site-specifically and would have lower affinity for DcR3. We prepared phage libraries expressing LIGHT mutants in which all the lysine residues were replaced with other amino acids. A lysine-deficient LIGHT mutant [mLIGHT-Lys(-)] was isolated by panning against lymphotoxin beta receptor (LTbetaR). mLIGHT-Lys(-) could be site-specifically PEGylated at its NH(2)-terminus, yielding molecular uniformity and in vitro bioactivity equal to that of non-PEGylated, wild-type LIGHT. Furthermore, mLIGHT-Lys(-) was not trapped by the nonfunctional DcR3, despite binding to its functional receptors. These results suggest that mLIGHT-Lys(-) might be a useful candidate for cancer therapy.

Comparison of the anti-tumor activity of native, secreted, and membrane-bound LIGHT in mouse tumor models

The TNF superfamily member LIGHT has potent anti-tumor activity through direct cytotoxicity and activation of the immune response, and is a promising candidate for cancer therapy. Natively, LIGHT exists as both a membrane-anchored form and a proteolytically processed, secreted form. However, the strength of the anti-tumor activity of each form of LIGHT has not been well defined. Here, to identify the optimal form of LIGHT for cancer gene therapy, we constructed fiber-mutant adenovirus vectors (AdRGD) encoding native full-length LIGHT (LIGHT/FL), stably membrane-anchored LIGHT (LIGHT/mem), and fully secreted LIGHT (LIGHT/sec). We then compared the anti-tumor effects of the different forms of LIGHT in mice by intratumoral injection of each AdRGD. We demonstrated that intratumoral injection of AdRGD-LIGHT/sec provided greater tumor suppression than AdRGD-LIGHT/FL, although this effect did not reach statistical significance. By comparison, AdRGD-LIGHT/mem had negligible anti-tumor activity. We also demonstrated that more CD4+ and CD8+ T cells accumulated inside tumors treated in vivo with AdRGD-LIGHT/sec than in tumors treated with AdRGD-LIGHT/FL or AdRGD-LIGHT/mem. These results suggest that the secreted form of LIGHT might be the optimal form for cancer gene therapy.