Alpha-crystallin B chains enhance cell migration in basal-like 2 triple-negative breast cancer cells.

Triple-negative breast cancer (TNBC) can be divided into six subtypes. Among these subtypes, the basal-like 2 (BL2) subtype shows the lowest five-year survival rate and highest risk of metastasis. Alpha-crystallin B chains (αB-crystallin), a small heat shock protein that is known to be involved in breast cancer metastasis, is highly expressed in the basal-like subtype but not in the other non-basal subtypes. Thus, we hypothesized that αB-crystallin may be an important factor involved in the worse prognosis of the BL2 subtype compared with those of the other TNBC subtypes. Here, we examined the role of αB-crystallin in cell motility in two TNBC cell lines: HCC1806 (BL2 subtype) and, as control, MDA-MB-436 (mesenchymal stem-like subtype). HCC1806 showed greater cell migration capacity and a higher expression level of the gene encoding αB-crystallin (CRYAB) than did MDA-MB-436. Short interfering RNA-mediated silencing of CRYAB expression significantly reduced the cell migration capacity of HCC1806 cells, whereas it had no effect in MDA-MB-436 cells, indicating that αB-crystallin is essential for the migration of HCC1806 cells. Thus, high αB-crystallin expression may be a contributing factor to the poor prognosis of BL2 TNBC.

Progesterone receptor membrane component 2 expression leads to erlotinib resistance in lung adenocarcinoma cells.

Epidermal growth factor receptor tyrosine kinase inhibitors (EGFR-TKIs) provide a favorable treatment outcome in patients with EGFR mutation-positive non-small cell lung cancer. However, most of such patients become resistant to EGFR-TKIs within a year. Thus, clarifying the mechanism of acquired resistance to EGFR-TKIs has been a research focus. Here, we demonstrated that the expression of progesterone receptor membrane component 2 (PGRMC2) was upregulated in an erlotinib-resistant cell line, PC9/ER, compared with the parental PC9 lung cancer cells. Our previous study showed that PGRMC1 is responsible for acquired resistance to erlotinib; however, PGRMC2 has not been discussed yet. Thus, the aim of this study was to determine the role of PGRMC2 in acquired resistance to erlotinib. Transfection with PGRMC2 siRNA significantly enhanced the sensitivity to erlotinib in PC9/ER cells. Furthermore, knockdown of PGRMC2 reduced the expression of p21, which is known as cell-cycle inhibitor and antiproliferative effector. These results suggest that PGRMC2 partially contributes to erlotinib resistance in PC9/ER cells, and that investigation into the effect of PGRMC2 on apoptosis and the cell cycle are warranted.

Ephrin type-A receptor 2 on tumor-derived exosomes enhances angiogenesis through the activation of MAPK signaling.

Exosomes are potent players in the development of metastases and they play an important role in cancer angiogenesis and exacerbation. However, it is unclear how proteins on exosomes affect development of blood vessel networks. In this study, we focused on relationships between membrane proteins on exosomes and angiogenesis using human umbilical vein endothelial cells (HUVEC). Lung tumor cell-derived exosomes induced tube formation and growth of endothelial cells in vitro in a dose-dependent manner involving MAPK activation, but this was not seen in normal lung epithelial cells. Ephrin type-A receptor 2 (EphA2) was identified by proteomic analysis and an inhibition assays showed it is a major MAPK activator on exosomes. Thus EphA2 on exosomes participates in angiogenesis as a ligand of the ephrin signaling pathway. These results support the development of novel therapeutic strategies such as blockade of remote cancer communications through exosomes.

Generation of a sensitive TNFR2-specific murine assays system.

Tumor necrosis factor (TNF)/TNF receptors (TNFR1/TNFR2) are considered to be potential drug targets to treat refractory diseases, including autoimmune diseases and malignant tumors. However, their specific functions, especially in the case of TNFR2, are poorly understood. In this study, we constructed a mouse TNFR2 (mTNFR2)-mediated biological assay system that shows no effects of mouse TNFR1 (mTNFR1) in order to screen mTNFR2-selective stimulating agents. Mouse TNFR1(-/-)R2(-/-) preadipocytes were transfected with the gene encoding the mTNFR2/mouse Fas (mFas) chimeric receptor in which the extracellular and transmembrane domains of mTNFR2 were fused to the intracellular domain of mFas. Our results demonstrated that this cell line exhibits highly sensitive mTNFR2-mediated cytotoxic effects. We propose that this mTNFR2-mediated biological assay system would be a useful tool to screen for mTNFR2-selective stimulating agents.

High-dose cutaneous exposure to mite allergen induces IgG-mediated protection against anaphylaxis.

BACKGROUND: The relationship among natural allergen exposure, induction of blocking antibody and the occurrence of atopic allergy-particularly in the presence of IgE production-is debatable. OBJECTIVE: To clarify the relationship between the dose of cutaneous exposure to dust mite allergen and susceptibility to the IgE-mediated allergic response in relation to IgG production. METHODS: NC/Nga mice were epicutaneously exposed to various doses of Dermatophagoides pteronyssinus allergen to induce atopic dermatitis-like skin lesions. We then evaluated the skin lesions, induction of mite-specific immune responses, and susceptibility to anaphylaxis. RESULTS: Dose-dependent exacerbation of atopic dermatitis-like skin lesions and increases in mite-specific IgG and IgE production were observed. However, mice exposed to relatively low doses of mite allergen showed hypersusceptibility to mite allergen-specific anaphylaxis. We also showed that adoptive transfer of total IgG from Dp-sensitized mice rescued mice from the hypersusceptibility seen in those exposed to low doses of mite allergen. CONCLUSIONS AND CLINICAL RELEVANCE: High-dose cutaneous exposure to dust mites induced effective blocking IgG production, even if accompanied by IgE production. Our data might support the concept that an increase in IgG titre, not a decrease in IgE titre, is a marker of clinical improvement in allergen-specific immunotherapy.

Porphyromonas gingivalis RagB is a proinflammatory signal transducer and activator of transcription 4 agonist.

Periodontal diseases are semi-ubiquitous and caused by chronic, plaque-induced inflammation. The 55-kDa immunodominant RagB outer membrane protein of Porphyromonas gingivalis, a keystone periodontal pathogen, has been proposed to facilitate nutrient transport. However, potential interactions between RagB and the innate response have not been examined. We determined that RagB exposure led to the differential and dose-related expression of multiple genes encoding proinflammatory mediators [interleukin-1α (IL-1α), IL-1ß, IL-6, IL-8 and CCL2; all P < 0.05] in primary human monocytes and to the secretion of tumor necrosis factor and IL-8, but not interferon-γ or IL-12. RagB was shown to be a Toll-like receptor 2 (TLR2) and TLR4 agonist that activated signal transducer and activator of transcription 4 and nuclear factor-κB signaling, as determined by a combination of blocking antibodies, pharmaceutical inhibitors and gene silencing. In keeping, a ΔragB mutant similarly exhibited reduced inflammatory capacity, which was rescued by ragB complementation. These results suggest that RagB elicits a major pro-inflammatory response in primary human monocytes and, therefore, could play an important role in the etiology of periodontitis and systemic sequelae.

A case of maxillary sarcoma in a chimpanzee (Pan troglodytes).

Oral malignancy is rare in chimpanzees. A 34-year-old female chimpanzee (Pan troglodytes) at Kumamoto Sanctuary, Japan, had developed it. Treatment is technically difficult for chimpanzees while malignant neoplasm is seemingly rising in captive populations. Widespread expert discussion, guidelines for treatment, especially for great apes in terminal stages is urgently needed.

Biochemical and hematologic effects of polyvinylpyrrolidone-wrapped fullerene C60 after oral administration.

The fullerene C60 is used in consumer products such as cosmetics owing to its antioxidative effects and is being developed for nanomedical applications. However, knowledge regarding the safety of fullerene C60, especially after oral administration, is sparse. Here, we examined the safety of fullerene C60 in mice after 7 d of exposure to orally administered polyvinylpyrrolidone (PVP)-wrapped fullerene C60 (PVP-fullerene C60). Mice treated with PVP-fullerene C60 showed few changes in the plasma levels of various markers of kidney and liver injury and experienced no significant hematologic effects. Furthermore, the histology of the colon of PVP-fullerene C60-treated mice was indistinguishable from that of control mice. These results suggest that PVP-fullerene C60 lacks toxicity after high-dose oral administration and indicate that PVP-fullerene C60 can be considered safe for oral medication. These data provide basic information that likely will facilitate the production of safe and effective forms of fullerene C60.

Epidermal growth factor receptor localized to exosome membranes as a possible biomarker for lung cancer diagnosis.

Detection of drug-target proteins and biomarkers that are expressed in cancer tissue has significant potential for both diagnosis and treatment of cancer. However, current immuno-histochemical and cytogenetic analyses of biopsy specimens for pre-operational diagnosis are highly invasive and often difficult to apply to lung cancer patients. The purpose of this study was to evaluate the possible utility of determining epidermal growth factor receptor (EGFR) expression on exosomal membranes using a targeted ELISA with an anti-CD81 antibody as a capture antibody for lung cancer diagnosis. While soluble EGFR (sEGFR) levels in plasma were not remarkably different between lung cancer patients and normal controls, significantly higher exosomal EGFR expression levels were observed in 5/9 cancer cases compared to normal controls. These results suggest that measurement of exosomal protein levels could be useful for in vitro diagnosis, and that exosomal EGFR is a possible biomarker for characterization of lung cancer.

Rho GDP-dissociation inhibitor alpha is associated with cancer metastasis in colon and prostate cancer.

Since metastasis is one of the most important prognostic factors in colorectal cancer, development of new methods to diagnose and prevent metastasis is highly desirable. However, the molecular mechanisms leading to the metastatic phenotype have not been well elucidated. In this study, a proteomics-based search was carried out for metastasis-related proteins in colorectal cancer by analyzing the differential expression of proteins in primary versus metastasis focus-derived colorectal tumor cells. Protein expression profiles were determined using a tissue microarray (TMA), and the results identified Rho GDP-dissociation inhibitor alpha (Rho GDI) as a metastasis-related protein in colon and prostate cancer patients. Consequently, Rho GDI may be useful as a diagnostic biomarker and/or a therapeutic to prevent colon and prostate cancer metastasis.

Arsenic trioxide induces down-regulation of gp46 via protein oxidation: proteomics analysis of oxidative modified proteins in As2O3-treated HTLV-1-infected cells.

Adult T-cell leukemia (ATL) is a severe chemotherapy-resistant malignancy associated with prolonged infection by the human T cell-lymphotropic virus 1 (HTLV-1) retrovirus. Epidemiology studies strongly indicate that an increase in HTLV-1 virus load is an important factor during the onset of ATL. Therefore, inhibition of the growth/transmission of HTLV-1 infected cells is a promising strategy in preventing the disease. In our previous study, we revealed that arsenic trioxide (As2O3), a drug used to treat acute promyelocytic leukemia (APL), exerts an inhibitory effect on syncytium formation between HTLV-1 infected cells and HeLa cells via suppression of HTLV-1 envelope protein gp46 expression at low concentrations. In this study, we analyze the mechanism of action of As2O3 using a proteomics approach. Our results suggest that down-regulation of gp46 might be related to As2O3-induced oxidation of the 71-kDa heat shock cognate protein (HSC70) and the 78-kDa glucose-regulated protein (BiP/GRP78). We postulate that AS2O3 exerts an inhibitory effect on HTLV-1 virus transmission via down-regulation of gp46-production, which might be caused by oxidative modification of various proteins such as chaperones.

Mutant TNF-alpha, mTNF-K90R, is a novel candidate adjuvant for a mucosal vaccine against HIV.

The development of a safe and effective mucosal vaccine adjuvant is a crucial step for the development of vaccines against human immunodeficiency virus type-1 (HIV). We have previously reported that a mutant tumor necrosis factor-alpha (TNF-alpha), mTNF-K90R, possessed strong mucosal vaccine adjuvant activities in mice. Here, we evaluated the potential of mTNF-K90R as a mucosal vaccine adjuvant for the induction of systemic and mucosal immune responses against HIV. Nasal immunization of BALB/c mice with 5 microg of an HIV gp120 env protein immunogen together with mTNF-K90R induced higher serum anti-HIV gp120 protein immunoglobulin G (IgG) responses than gp120 alone. Furthermore, mTNF-K90R induced anti-gp120 IgA responses in nasal as well as vaginal washes from immunized mice, although these were not administration sites. Again, responses with mTNF-K90R were higher than with gp120 alone. These results indicate that mTNF-K90R may be applicable as amucosal adjuvant for HIV vaccination to induce both systemic and mucosal immune responses.

Creation of an improved mutant TNF with TNFR1-selectivity and antagonistic activity by phage display technology.

Tumor necrosis factor-alpha (TNF), which binds two types of TNF receptors (TNFR1 and TNFR2), regulates the onset and exacerbation of autoimmune diseases such as rheumatoid arthritis and Crohn's disease. In particular, TNFR1-mediated signals are predominantly related to the induction of inflammatory responses. We have previously generated a TNFR1-selective antagonistic TNF-mutant (mutTNF) and shown that mutTNF efficiently inhibits TNFR1-mediated bioactivity in vitro and attenuates inflammatory conditions in vivo. In this study, we aimed to improve the TNFR1-selectivity of mutTNF This was achieved by constructing a phage library displaying mutTNF-based variants, in which the amino acid residues at the predicted receptor binding sites were substituted to other amino acids. From this mutant TNF library, 20 candidate TNFR1-selective antagonists were isolated. Like mutTNF, all 20 candidates were found to have an inhibitory effect on TNFR1-mediated bioactivity. However, one of the mutants, N7, displayed significantly more than 40-fold greater TNFR1-selectivty than mutTNF. Therefore, N7 could be a promising anti-autoimmune agent that does not interfere with TNFR2-mediated signaling pathways.

Rapid isolation of intrabody candidates by using an optimized non-immune phage antibody library.

Phage antibody library is a promising tool for rapidly creating in vitro single-chain Fv (scFv) antibodies to various antigens. The scFv can also act like a subcellularly-expressed antibody, known as intrabody, and can either be used as a novel research tool or used efficiently for targeted molecular therapy. However, there are only a few existing reports about the successful expression of scFvs as functional antibodies in the cell, mainly because poor quality scFv phage antibody libraries were used to isolate the intrabody clones. The aim of this study was to isolate intrabody-forming scFv clones from the nonimmune scFv phage antibody library we have generated. Using this library, we isolated a scFv clone against the apoptosis-related intracellular protein Bid in two weeks. To evaluate the intrabody-forming quality of this anti-Bid scFv clone, we expressed it in cultured mammalian cells after fusing it with the fluorescent protein Venus. The expression of the soluble form of anti-Bid scFv-Venus fusion protein was confirmed by fluorescence microscopy analysis. These results show that our scFv phage library is not only optimized for antibody production but can also be used to efficiently generate intrabodies.

The specific effect of 2-methoxyestradiol on lymphatic vascular endothelial cells.

Lymphatic metastasis of tumors is one of the most important prognostic factors and provides valuable information for decisions on appropriate surgical protocols. Recent studies have demonstrated that lymphangiogenesis of lymphatic vascular endothelial cells into tumors is a key event in lymphatic metastasis. Therefore, control of lymphangiogenesis is a promising strategy for treatment or prevention of tumor metastasis and lymphatic disorders. However, mechanisms of lymphangiogenesis or its specific inhibition are not well-understood. In this study we examined effects of various types of signaling inhibitors on tube formation in human lymphatic microvascular endothelial cells (LECs) and blood microvascular endothelial cells (BECs) in vitro. We found that tube formation of LECs was specifically inhibited by 2-methoxyestradiol (2ME). This observation is of potential benefit in understanding the molecular mechanism of lymphangiogenesis. Furthermore, 2ME could therefore offer specific protection against lymphatic metastasis and lymphangiogenesis-related diseases.

Effect of protein properties on display efficiency using the M13 phage display system.

The M13 phage display system is a powerful technology for engineering proteins such as functional mutant proteins and peptides. In this system, it is necessary that the protein is displayed on the phage surface. Therefore, its application is often limited when a protein is poorly displayed. In this study, we attempted to understand the relationship between a protein's properties and its display efficiency using the well-known pIII and pVIII type phage display system. The display of positively charged SV40 NLS and HIV-1 Tat peptides on pill was less efficient than that of the neutrally charged RGDS peptide. When different molecular weight proteins (1.5-58 kDa) were displayed on pIII and pVIII, their display efficiencies were directly influenced by their molecular weights. These results indicate the usefulness in predicting a desired protein's compatibility with protein and peptide engineering using the phage display system.

Comparative study on transduction and toxicity of protein transduction domains.

BACKGROUND AND PURPOSE: Protein transduction domains (PTDs), such as Tat, antennapedia homeoprotein (Antp), Rev and VP22, have been extensively utilized for intracellular delivery of biologically active macromolecules in vitro and in vivo. There is little known, however, about the relative transduction efficacy, cytotoxicity and internalization mechanism of individual PTDs. EXPERIMENTAL APPROACH: We examined the cargo delivery efficacies of four major PTDs (Tat, Antp, Rev and VP22) and evaluated their toxicities and cell internalizing pathways in various cell lines. KEY RESULTS: The relative order of the transduction efficacy of these PTDs conjugated to fluorescein was Rev>Antp>Tat>VP22, independent of cell type (HeLa, HaCaT, A431, Jurkat, MOLT-4 and HL60 cells). Antp produced significant toxicity in HeLa and Jurkat cells, and Rev produced significant toxicity in Jurkat cells. Flow cytometric analysis demonstrated that the uptake of PTD-fluorescein conjugate was dose-dependently inhibited by methyl-beta-cyclodextrin, cytochalasin D and amiloride, indicating that all four PTDs were internalized by the macropinocytotic pathway. Accordingly, in cells co-treated with 'Tat-fused' endosome-disruptive HA2 peptides (HA2-Tat) and independent PTD-fluorescent protein conjugates, fluorescence spread throughout the cytosol, indicating that all four PTDs were internalized into the same vesicles as Tat. CONCLUSIONS AND IMPLICATIONS: These findings suggest that macropinocytosis-dependent internalization is a crucial step in PTD-mediated molecular transduction. From the viewpoint of developing effective and safe protein transduction technology, although Tat was the most versatile carrier among the peptides studied, PTDs should be selected based on their individual characteristics.

Creation of a novel cell penetrating peptide, using a random 18mer peptides library.

Cell penetrating peptides (CPPs) have drawn attention as carriers for intracellular drug delivery. It is commonly believed that TAT peptide is the best carrier among the existing CPPs due to its high translocational activity. Despite considerable research, the cellular uptake mechanism of TAT peptide remains unclear. Additionally, the transduction efficiency of TAT peptide is insufficient for use in intracellular therapy. In this study, we attempted to identify novel CPPs from a random 18mer peptide library using a phage display system. To isolate novel CPPs more effectively, PSIF (protein synthesis inhibition factor) was used with the screening system. Consequently, we isolated 7 novel CPPs from the library and determined by flow cytometry and confocal laser microscopy that these CPPs were taken up into cells. Once the cellular uptake pathway of these CPPs has been determined, it may be possible to use them for intracellular therapy.

Further evidence that major outer membrane proteins homologous to OmpA in Porphyromonas gingivalis stabilize bacterial cells.

INTRODUCTION: Porphyromonas gingivalis is one of the most important bacteria in the progression of chronic periodontal disease. We hypothesized that the major outer membrane proteins Pgm6/7, which are homologous to the OmpA protein in Escherichia coli, might contribute to the stabilization of the cell surface. In this study, the effects of Pgm6/7 on the cell surface were examined morphologically. METHODS: Deletion mutants of Pgm6/7 (Delta694, Delta695 and Delta695-694) were constructed using the polymerase chain reaction-based overlap extension method. Wild-type ATCC 33277 and Pgm6/7 mutants were grown under anaerobic conditions. Whole cells and thin sections of fixed cells were stained and examined by transmission electron microscopy. RESULTS: Compared with the wild-type, numerous vesicles released from cells were observed in each deletion mutant. The outer membrane appeared wavy and irregular. Increased numbers of vesicles were confirmed after their preparation from the culture supernatant. Total gingipain activity in vesicles was increased five- to 10-fold in the deletion mutants. CONCLUSION: This report provides further evidence that Pgm6/7 proteins in P. gingivalis play an important role in the maintenance of bacterial outer membrane integrity.

Optimization of anti-tumor necrosis factor-alpha single chain Fv displayed on phages for creation of functional antibodies.

In this study, we converted the immunoglobulin-type anti-human tumor necrosis factor-alpha (TNF-alpha) monoclonal antibody (Mab) to a scFv-type antibody in order to assess its basic properties. The immunoglobulin VH and VL genes were isolated from the hybridoma that produced an anti-TNF-alpha neutralizing Mab, and they were then linked together to create scFvs of the VL-VH or VH-VL-form. The binding affinity to TNF-a was retained in both scFvs. Interestingly, the VL-VH-type scFv effectively inhibited the TNF-alpha-mediated cytotoxicity, while this neutralization activity was dramatically decreased in the VH-VL-type scFv. These results suggest that the VL-VH-type scFv is a suitable template to create improved versions of the anti-TNF-alpha antibody using a phage display system, and they also show that the structural format must be taken into account in manufacturing scFvs.