Yasuhiro Omata

Non-equilibrium atmospheric pressure plasmas modulate cell cycle-related gene expressions in melanocytic tumors of RET-transgenic mice

The incidence of cutaneous malignant melanoma is increasing at a greater rate than that of any other cancer in the world. However, an effective therapy for malignant melanoma has not been established. Recently, some studies have shown an antitumor effect of non‐equilibrium atmospheric pressure plasmas (NEAPPs) in vitro. Here, we examined the in vivo effect of NEAPP on cell cycle regulators, key elements for malignant transformation, in spontaneously developed benign melanocytic tumors in a hairless animal model. NEAPP irradiation decreased expression levels of cell cycle promoters, Cyclin D1, E1 and E2, and increased expression level of a cell cycle repressor, p27KIP1. Cyclin D1, E1 and E2 and p27KIP expression levels were associated with malignant transformation of the benign tumor in the animal model. Our results suggest that NEAPP irradiation suppresses malignant transformation of a benign melanocytic tumor via control of the expression levels of cell cycle regulators.

Arsenite-Mediated Promotion of Anchorage-Independent Growth of HaCaT Cells through Placental Growth Factor

Various cancers including skin cancer are increasing in 45 million people exposed to arsenic above the World Health Organizations guideline value of 10 μg l(-1). However, there is limited information on key molecules regulating arsenic-mediated carcinogenesis. Our fieldwork in Bangladesh demonstrated that levels of placental growth factor (PlGF) in urine samples from residents of cancer-prone areas with arsenic-polluted drinking water were higher than those in urine samples from residents of an area that was not polluted with arsenic. Our experimental study in human nontumorigenic HaCaT skin keratinocytes showed that arsenite promoted anchorage-independent growth with increased expression and secretion of PlGF, a ligand of vascular endothelial growth factor receptor1 (VEGFR1), and increased VEGFR1/mitogen-activated protein kinase/ERK kinase (MEK)/extracellular signal-regulated kinase (ERK) activities. The arsenite-mediated promotion of anchorage-independent growth was strongly inhibited by PlGF depletion with decreased activities of the PlGF/VEGFR1/MEK/ERK pathway. Moreover, arsenite proteasome-dependently degrades metal-regulatory transcription factor-1 (MTF-1) protein, resulting in a decreased amount of MTF-1 protein binding to the PlGF promoter. MTF-1 negatively controlled PlGF transcription in HaCaT cells, resulting in increased PlGF transcription. These results suggest that arsenite-mediated MTF-1 degradation enhances the activity of PlGF/VEGFR1/MEK/ERK signaling, resulting in promotion of the malignant transformation of keratinocytes. Thus, this study proposed a molecular mechanism for arsenite-mediated development of skin cancer.

Non-thermal atmospheric pressure plasmas as a novel candidate for preventive therapy of melanoma

Due to the increased ultraviolet radiation, the incidence of melanoma is increasing worldwide more than that of any other cancer. In this study, the effects of irradiation of non-thermal atmospheric pressure plasmas (NEAPPs) on benign melanocytic tumors from our original hairless model mice (HL-RET-mice), in which benign melanocytic tumors and melanomas spontaneously develop in the skin stepwise, were examined. Expression levels of melanoma cell adhesion molecule (MCAM) and matrix metalloproteinase-2 (MMP-2) mRNA in melanomas were higher than those in benign melanocytic tumors in the mice. Repeated irradiation of non-thermal atmospheric pressure plasmas (NEAPPs) for the benign tumors decreased the expression levels of MCAM and MMP-2 mRNA in the tumors from the mice. Previous studies showed that MCAM sites are upstream of MMP-2, that MCAM regulates transcription of MMP-2 in melanoma cells and that MMP-2 is associated with the conversion of a benign tumor to a malignant tumor. Therefore, our results suggest that the NEAPP irradiation-mediated decrease in the expression level of MMP-2 in benign melanocytic tumors is associated with decreased expression levels of MCAM. Moreover, NEAPP irradiation might be a potential candidate for therapy to prevent melanoma development through suppression of malignant conversion in benign melanocytic tumors.

Nontoxic singlet oxygen generator as a therapeutic candidate for treating tauopathies

Methylene blue (MB) inhibits the aggregation of tau, a main constituent of neurofibrillary tangles. However, MB’s mode of action in vivo is not fully understood. MB treatment reduced the amount of sarkosyl-insoluble tau in Drosophila that express human wild-type tau. MB concurrently ameliorated the climbing deficits of transgenic tau flies to a limited extent and diminished the climbing activity of wild-type flies. MB also decreased the survival rate of wild-type flies. Based on its photosensitive efficacies, we surmised that singlet oxygen generated through MB under light might contribute to both the beneficial and toxic effects of MB in vivo. We identified rose bengal (RB) that suppressed tau accumulation and ameliorated the behavioral deficits to a lesser extent than MB. Unlike MB, RB did not reduce the survival rate of flies. Our findings indicate that singlet oxygen generators with little toxicity may be suitable drug candidates for treating tauopathies.

Oral exposure to arsenic causes hearing loss in young people aged 12–29 years and in young mice

There is no information on the association between oral exposure to arsenic (As) and hearing loss in humans or mice. In this combined epidemiological study and experimental study, the association of oral exposure to As with hearing loss in people aged 12–29 years and young mice was examined. Subjects in the exposure group (n = 48), who were drinking tube well water contaminated with As, showed significantly higher risks of hearing loss at 4 kHz [odds ratio (OR) = 7.60; 95% confidence interval (CI): 1.56, 57.88], 8 kHz (OR = 5.00; 95% CI: 1.48, 18.90) and 12 kHz (OR = 8.72; 95% CI: 2.09, 47.77) than did subjects in the control group (n = 29). We next performed an experiment in which young mice were exposed to As via drinking water at 22.5 mg/L, which is a much greater concentration than that in human studies. The exposure group showed hearing loss and accumulation of As in inner ears. Ex vivo exposure of the organ of Corti from mice exposed to As significantly decreased the number of auditory neurons and fibers. Thus, our combined study showed that oral exposure to As caused hearing loss in young people and young mice.

Commentary to Pastore et al. (2014): Epidermal growth factor receptor signaling in keratinocyte biology: implications for skin toxicity of tyrosine kinase inhibitors

models to mimic these toxic reactions would provide mechanism-based treatment or prevention of TKIs-related skin diseases. Tyrosine kinases are important molecules in biological processes including growth, differentiation, and metabolism, and also in several steps of neoplastic development and progression. TKIs have therefore received much attention as targets of molecular-targeted therapy of cancer. However, their toxicity has not been well understood. This review is important because it focuses on the molecular mechanisms underlying the toxicity of TKIs. There is great potential for cross-reactivities with most TKIs. This is because almost all TKIs interfere with enzyme activity of the intracellular kinase domain by competing with the ATP binding site, and there is little variation in the highly conserved ATP binding module of all protein kinase structures (Knight et al. 2007). Pastore et al. stated that the combined inhibition of three receptors, PDGFR, VEGFR, and c-KIT, could be pathogenetically involved in foot-and-hand skin reactions. Selectivity of TKIs for only kinases involved in tumor pathogenesis is very important to prevent adverse effects. It is known that the catalytic activity of protein tyrosine kinases (PTKs) is upregulated by major autophosphorylation sites (Kawamoto et al. 2004). c-RET, a representative receptor type of PTKs, is expressed in skin (Adly et al. 2008) and is correlated with skin melanoma in humans (Narita et al. 2009; Ohshima et al. 2010). We previously demonstrated that a specific cysteine residue (Cys376 of extracellular domain-deleted mutant RET-PTC-1, an equivalent of Cys987 of c-RET) in the alpha-helix H region of the catalytic domain is crucially involved in the maintenance and upregulation of these kinases activities (Kato et al. 2000; Takeda et al. 2001, 2006) as well as the known tyrosine phosphorylation-dependent activation mechanism. Pastore et al. (Arch Toxicol 88(1): 1189–1203, 2014) recently reviewed the homoeostatic mechanisms of skin controlled by epidermal growth factor receptor (EGFR) and the influence of EGFR inhibition on the mechanisms. Tyrosine kinase inhibitors (TKIs) have increased survival of patients compared to conventional chemotherapy drugs. However, TKIs, especially EGFR inhibitors, are frequently associated with adverse effects on the skin. Pastore et al. discussed the molecular mechanisms of TKI-associated dermatologic toxic events. EGFR has a central role in the distinct situation of skin homoeostasis, including survival and proliferation of keratinocytes, epidermal barrier function, and regulation of immune processes occurring in the skin. TKIs perturb skin homeostasis by inhibiting these functions of EGFR and consequently induce adverse effects on the skin. These drugs have not been sufficiently investigated for their toxicological profile. Pastore et al. introduced recent studies suggesting possible therapeutic strategies to suppress skin inflammation induced by inhibition of EGFR. For instance, elimination of skin monocytes (Mascia et al. 2013) or inhibition of mast cells (Lichtenberger et al. 2013) partially improved the skin inflammation of keratinocyte-restricted EGFR knockout mice. They indicated that combining the research of these side effects in humans at the cellular and molecular levels with animal

A disadvantageous effect of adsorption of barium by melanin on transforming activity

At present, beneficial effects of melanin and harmful effects of barium have been reported. However, little is known about the adsorption of barium, and even less is known about the biological significance of adsorption of barium by melanin. In this study, we showed that there was a strong correlation between the digitalized level of skin pigmentation and barium level in murine skin compared to the correlations between skin pigmentation level and levels of homologous elements of barium (magnesium, calcium and strontium). The concentration of subcutaneously injected barium in skin with a high level of pigmentation was higher than that in skin with a low level of pigmentation. Our cell-free experiment using the Langmuir isotherm for adsorption of barium in synthetic melanin also provided direct evidence of adsorption of barium by melanin. We then investigated the biological significance of melanin-mediated barium adsorption. We found barium-mediated increase in transforming activity in pigmented melanocytes (melan-a) but not in unpigmented melanocytes (melan-c) after confirming that the barium level in melan-a melanocytes was 3.4-fold higher than that in melan-c melanocytes after culture of 5 μM barium for 24 h. Taken together, our results not only indicate adsorption of barium by melanin in mice, cells and cell-free systems but also suggest a disadvantageous effect of adsorption of barium by melanin on transforming activity in cultured cells.

Pyroglutamate–amyloid-β peptide expression in Drosophila leads to caspase-dependent and endoplasmic reticulum stress–related progressive neurodegeneration

Alzheimers disease (AD) is the most common neurodegenerative disorder among the elderly. During the progression of AD, massive neuronal degeneration occurs in the late stage of the disease; however, the molecular mechanisms responsible for this neuronal loss remain unknown. AβpE3-42 (an N-terminal-truncated amyloid-β peptide that begins with pyroglutamate at the third position) is produced during late-stage AD. It also aggregates more rapidly in vitro and exhibits greater toxicity in neurons than full-length Aβ1-42. In the present study, we established a Drosophila melanogaster model that expresses Aβ3-42E3Q, which effectively produces AβpE3-42, and investigated the function of AβpE3-42 using the photoreceptor neurons of Drosophila. AβpE3-42 induced caspase-dependent apoptosis and caused progressive degeneration in photoreceptor neurons. Mutations in ER stress response genes or the administration of an inhibitor of the ER stress response markedly suppressed the degeneration phenotype, suggesting that the ER stress response plays an important role in neurodegeneration caused by AβpE3-42. We also confirmed that human Tau-dependent apoptotic induction was strongly enhanced by AβpE3-42. Thus, AβpE3-42 expression system in the fly may be a promising new tool for studying late-onset neurodegeneration in AD.

[Analysis of heavy-metal-mediated disease and development of a novel remediation system based on fieldwork and experimental research].

Heavy-metal pollution occurs in various environments, including water, air and soil, and has serious effects on human health. Since heavy-metal pollution in drinking water causes various diseases including skin cancer, it has become a global problem worldwide. However, there is limited information on the mechanism of development of heavy-metal-mediated disease. We performed both fieldwork and experimental studies to elucidate the levels of heavy-metal pollution and mechanisms of development of heavy-metal-related disease and to develop a novel remediation system. Our fieldwork in Bangladesh, Vietnam and Malaysia demonstrated that drinking well water in these countries was polluted with high concentrations of several heavy metals including arsenic, barium, iron and manganese. Our experimental studies based on the data from our fieldwork demonstrated that these heavy metals caused skin cancer and hearing loss. Further experimental studies resulted in the development of a novel remediation system with which toxic heavy metals were absorbed from polluted drinking water. Implementation of both fieldwork and experimental studies is important for prediction, prevention and therapy of heavy-metal-mediated diseases.

[Development of Preventive Therapy by Clarification of Mechanisms of Environmental-Factor-Mediated Diseases].

Environmental factors affecting human health are generally classified into physical, chemical and biological factors. In this review article, we focus on ultraviolet (UV) as a physical factor, heavy metals as a chemical factor and Japanese cedar pollens as a biological factor. Since we believe that progress based on both fieldwork research and experimental research is essential in hygiene study, we included the results of both the research approached. We first introduced the mechanism of development of and prevention of UV-mediated skin melanoma in our experimental research after showing our epidemiological research on UV-mediated DNA damage in humans. We then introduced our evaluation of toxicity and development of a remediation system in our experimental research on heavy metals after showing our fieldwork research for the monitoring of drinking water from wells in Asian countries. We finally introduced the results of pathogenic analysis of pollinosis in our clinical study. We would be very happy if young researchers would re-realize the importance of experimental research as well as epidemiological research in hygiene study.