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.

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.

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.

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.

Promotion of apoptosis and cytochrome c depletion by a low-temperature environment in hindlimb-unloading rats.

OBJECTIVE: This study aimed to clarify the influence of a low-temperature environment on muscle atrophy and apoptosis. METHODS: Wistar rats were divided into four groups: two groups of hindlimb-unloading rats maintained in a normal (25°C, HU) or low-temperature (10°C, HU+LT) environment for 3 weeks and two corresponding control groups (CON; normal temperature, CON+LT; low-temperature). RESULTS: The soleus muscle wet weight and muscle-to-body mass ratio were lower in the experimental groups than in the control groups. The cross-sectional areas of myofibers in the HU+LT and HU groups were significantly decreased than those in the CON and CON+LT groups. Ubiquitin ladder levels from soleus muscle lysates were significantly increased in the HU+LT group. Caspase-3-activated myofibers were observed only in the HU+LT group. Decreased cytochrome c levels were present in these caspase-3-activated myofibers. Meanwhile, cytochrome c levels were increased significantly in CON+LT rats but unchanged in HU+LT rats. CONCLUSION: Our results suggest that apoptosis caused by hindlimb unloading at low temperatures is associated with a lack of cytochrome c in myofibers. This indicates that long-term hindlimb unloading at low temperatures did not suppress muscle atrophy. We conclude that low-temperature stimulation should not be used as a long-term treatment for preventing disuse atrophy.

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.

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.

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.

A norovirus outbreak associated with environmental contamination at a hotel.

SUMMARYIn December 2006, an outbreak of gastroenteritis occurred involving 372 guests and 72 employees at a hotel after a guest vomited in corridors on the third (F3) and 25th (F25) floors. Norovirus with identical genotype was confirmed by real-time reverse transcription-polymerase chain reaction in faecal samples from guest cases and employees. Spread of the outbreak on F25 was compared with that on F3. The attack rate in the guests who visited F25 alone (15·0%, 106/708 guests) was significantly higher than in those who visited F3 alone (3·5%, 163/4710 guests) (relative risk 4·3, 95% confidence interval 3·4-5·5, P < 0·001). The outbreak on F3 ended within 2 days, while that on F25 extended over 7 days. The environmental ratios of F3 to F25 were 7·4 for volume, 6·9 for floor area and 7·6 for ventilation rate. This outbreak suggests that environmental differences can affect the propagation and persistence of a norovirus outbreak following environmental contamination.

Size-dependent immune-modulating effect of amorphous nanosilica particles.

The immune-modulating effect following intradermal injection of various-sized amorphous silica particles was analyzed in terms of induction of ovalbumin-specific CD8+ T cells in vivo. IFN-gamma ELISPOT assays revealed that only nanosilica particles with a diameter of less than 100 nm significantly enhanced CD8+ T cell responses against ovalbumin. These results indicate that the size of nanomaterials is a critical determinant in terms of their safe use.

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.

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.

Size-dependent cytotoxic effects of amorphous silica nanoparticles on Langerhans cells.

Amorphous silica nanoparticles (nSPs), are widely used in medicines, cosmetics and food. However, due to their reduced particle size they are suspected to pose new risks induced by changes in biological reactivity and kinetics, which differ from those of bulk materials. In a previous study, we showed that silica particles with a diameter of 70 nm penetrated the stratum corneum (SC) of mouse skin and were taken up by living cells such as keratinocytes and Langerhans cells. To clarify the relationship between particle size, distribution and cellular response, we have evaluated size-dependent intracellular localization and cytotoxicity of silica particles, using the mouse epidermal Langerhans cell line XS52. On treatment with silica particles of diameters 70, 300, and 1000 nm, cellular uptake and cytotoxicity increased with reduction in particle size. These results suggest that smaller sized silica particles induced greater cytotoxicity against Langerhans cells, which was correlated with the quantity of particle uptake into the cells.

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.

Tor-mediated induction of autophagy via an Apg1 protein kinase complex.

Autophagy is a membrane trafficking to vacuole/lysosome induced by nutrient starvation. In Saccharomyces cerevisiae, Tor protein, a phosphatidylinositol kinase-related kinase, is involved in the repression of autophagy induction by a largely unknown mechanism. Here, we show that the protein kinase activity of Apg1 is enhanced by starvation or rapamycin treatment. In addition, we have also found that Apg13, which binds to and activates Apg1, is hyperphosphorylated in a Tor-dependent manner, reducing its affinity to Apg1. This Apg1-Apg13 association is required for autophagy, but not for the cytoplasm-to-vacuole targeting (Cvt) pathway, another vesicular transport mechanism in which factors essential for autophagy (Apg proteins) are also employed under vegetative growth conditions. Finally, other Apg1-associating proteins, such as Apg17 and Cvt9, are shown to function specifically in autophagy or the Cvt pathway, respectively, suggesting that the Apg1 complex plays an important role in switching between two distinct vesicular transport systems in a nutrient-dependent manner.

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.

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.

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.