Tasuku Hirayama

A highly selective turn-on fluorescent probe for iron(II) to visualize labile iron in living cells

Although labile iron plays critical roles in diverse biological processes in living cells, the physiological and pathophysiological functions of iron have not been sufficiently explored, partially due to a lack of methods for visualizing intracellular labile iron. In this edge article, we present a novel turn-on fluorescent probe (RhoNox-1) for the selective detection of Fe2+ based on N-oxide chemistry. Spectroscopic studies combined with DFT calculations and electrochemical studies revealed that fluorescence quenching of RhoNox-1 occurred in physiological conditions, which was attributed to non-radiative deactivation of the excited state of tertiary amine N-oxide substituted xanthene involving a twisted internal charge transfer (TICT) process and partially due to photo-induced electron transfer (PET) from the N-oxide group. RhoNox-1 showed significant enhancement of the fluorescence signal in Fe2+-loaded cells via selective Fe2+-mediated deoxygenation of the N-oxide group and also successfully detected basal and endogenous labile Fe2+ in living cells.

Facile One-Pot Synthesis of (1, 2, 3)Triazolo(1, 5-a)Pyridines from 2- Acylpyridines by Copper(II)-Catalyzed Oxidative NN Bond Formation

An efficient and simple method for the synthesis of various [1, 2, 3]triazolo[1, 5-a]pyridines has been established. The method involves a copper(II)-catalyzed oxidative N-N bond formation that uses atmospheric oxygen as the terminal oxidant following hydrazonation in one pot. The use of ethyl acetate as the solvent dramatically promotes the oxidative N-N bond-formation reaction and enables the application of oxidative cyclization in the efficient one-pot reaction. A mechanism for the reaction was proposed on the basis of the results of a spectroscopic study.

Iron stimulates plasma-activated medium-induced A549 cell injury

Non-thermal atmospheric pressure plasma is applicable to living cells and has emerged as a novel technology for cancer therapy. Plasma has recently been shown to affect cells not only by direct irradiation, but also by indirect treatments with previously prepared plasma-activated medium (PAM). Iron is an indispensable element but is also potentially toxic because it generates the hydroxyl radical (•OH) in the presence of hydrogen peroxide (H2O2) via the Fenton reaction. The aim of the present study was to demonstrate the contribution of iron to PAM-induced A549 adenocarcinoma cell apoptosis. We detected the generation of •OH and elevation of intracellular ferrous ions in PAM-treated cells and found that they were inhibited by iron chelator. The elevations observed in ferrous ions may have been due to their release from the intracellular iron store, ferritin. Hydroxyl radical-induced DNA injury was followed by the activation of poly(ADP-ribose) polymerase-1, depletion of NAD+ and ATP, and elevations in intracellular Ca2+. The sensitivities of normal cells such as smooth muscle cells and keratinocytes to PAM were less than that of A549 cells. These results demonstrated that H2O2 in PAM and/or •OH generated in the presence of iron ions disturbed the mitochondrial-nuclear network in cancer cells.

Histological detection of catalytic ferrous iron with the selective turn-on fluorescent probe RhoNox-1 in a Fenton reaction-based rat renal carcinogenesis model

Abstract Iron overload of a chronic nature has been associated with a wide variety of human diseases, including infection, carcinogenesis, and atherosclerosis. Recently, a highly specific turn-on fluorescent probe (RhoNox-1) specific to labile ferrous iron [Fe(II)], but not to labile ferric iron [Fe(III)], was developed. The evaluation of Fe(II) is more important than Fe(III) in vivo in that Fe(II) is an initiating component of the Fenton reaction. In this study, we applied this probe to frozen sections of an established Fenton reaction-based rat renal carcinogenesis model with an iron chelate, ferric nitrilotriacetate (Fe-NTA), in which catalytic iron induces the Fenton reaction specifically in the renal proximal tubules, presumably after iron reduction. Notably, this probe reacted with Fe(II) but with neither Fe(II)-NTA, Fe(III) nor Fe(III)-NTA in vitro. Prominent red fluorescent color was explicitly observed in and around the lumina of renal proximal tubules 1 h after an intraperitoneal injection of 10–35 mg iron/kg Fe-NTA, which was dose-dependent, according to semiquantitative analysis. The RhoNox-1 signal colocalized with the generation of hydroxyl radicals, as detected by hydroxyphenyl fluorescein (HPF). The results demonstrate the transformation of Fe(III)-NTA to Fe(II) in vivo in the Fe-NTA-induced renal carcinogenesis model. Therefore, this probe would be useful for localizing catalytic Fe(II) in studies using tissues.

Chemical tools for detecting Fe ions

Owing to its distinctive electrochemical properties with interconvertible multiple oxidation states, iron plays a significant role in various physiologically important functions such as respiration, oxygen transport, energy production, and enzymatic reactions. This redox activity can also potentially produce cellular damage and death, and numerous diseases are related to iron overload resulting from the dysfunction of the iron regulatory system. In this case, “free iron” or “labile iron,” which refers to iron ion weakly bound or not bound to proteins, causes aberrant production of reactive oxygen species. With the aim of elucidating the variation of labile iron involved in pathological processes, some chemical tools that can qualitatively and/or quantitatively monitor iron have been utilized to investigate the distribution, accumulation, and flux of biological iron species. Since iron ions show unique reactivity depending on its redox state, i.e., Fe2+ or Fe3+ (or transiently higher oxidative states), methods for the separate detection of iron species with different redox states are preferred to understand its physiological and pathological roles more in detail. The scope of this review article covers from classical chromogenic to newly emerging chemical tools for the detection of Fe ions. In particular, chemical tools applicable to biological studies will be presented.

Role of hemoglobin and transferrin in multi-wall carbon nanotube-induced mesothelial injury and carcinogenesis

Multi‐wall carbon nanotubes (MWCNT) are a form of flexible fibrous nanomaterial with high electrical and thermal conductivity. However, 50‐nm MWCNT in diameter causes malignant mesothelioma (MM) in rodents and, thus, the International Agency of Research on Cancer has designated them as a possible human carcinogen. Little is known about the molecular mechanism through which MWCNT causes MM. To elucidate the carcinogenic mechanisms of MWCNT in mesothelial cells, we used a variety of lysates to comprehensively identify proteins specifically adsorbed on pristine MWCNT of different diameters (50 nm, NT50; 100 nm, NT100; 150 nm, NT150; and 15 nm/tangled, NTtngl) using mass spectrometry. We identified >400 proteins, which included hemoglobin, histone, transferrin and various proteins associated with oxidative stress, among which we selected hemoglobin and transferrin for coating MWCNT to further evaluate cytotoxicity, wound healing, intracellular catalytic ferrous iron and oxidative stress in rat peritoneal mesothelial cells (RPMC). Cytotoxicity to RPMC was observed with pristine NT50 but not with NTtngl. Coating NT50 with hemoglobin or transferrin significantly aggravated cytotoxicity to RPMC, with an increase in cellular catalytic ferrous iron and DNA damage also observed. Knockdown of transferrin receptor with ferristatin II decreased not only NT50 uptake but also cellular catalytic ferrous iron. Our results suggest that adsorption of hemoglobin and transferrin on the surface of NT50 play a role in causing mesothelial iron overload, contributing to oxidative damage and possibly subsequent carcinogenesis in mesothelial cells. Uptake of NT50 at least partially depends on transferrin receptor 1. Modifications of NT50 surface may decrease this human risk.

Contrasting intra- and extracellular distribution of catalytic ferrous iron in ovalbumin-induced peritonitis.

Iron is an essential nutrient for every type of life on earth. However, excess iron is cytotoxic and can lead to an increased cancer risk in humans. Catalytic ferrous iron [Fe(II)] is an initiator of the Fenton reaction, which causes oxidative stress by generating hydroxyl radicals. Recently, it became possible to localize catalytic Fe(II) in situ with a turn-on fluorescent probe, RhoNox-1. Here, we screened each organ/cell of rats to globally evaluate the distribution of catalytic Fe(II) and found that eosinophils showed the highest abundance. In various cells, lysosomes were the major organelle, sharing ∼40-80% of RhoNox-1 fluorescence. We then used an ovalbumin-induced allergic peritonitis model to study the dynamics of catalytic Fe(II). Peritoneal lavage revealed that the total iron contents per cell were significantly decreased, whereas an increase in the number of inflammatory cells (macrophages, neutrophils, eosinophils and lymphocytes) resulted in an increased total iron content of the peritoneal inflammatory cells. Notably, macrophages, eosinophils and neutrophils exhibited significantly increased catalytic Fe(II) with increased DMT1 expression and decreased ferritin expression, though catalytic Fe(II) was significantly decreased in the peritoneal lavage fluid. In conclusion, catalytic Fe(II) in situ more directly reflects cellular activity and the accompanying pathology than total iron does.

Ovarian endometriosis-associated stromal cells reveal persistently high affinity for iron

Ovarian endometriosis is a recognized risk for infertility and epithelial ovarian cancer, presumably due to iron overload resulting from repeated hemorrhage. To find a clue for early detection and prevention of ovarian endometriosis-associated cancer, it is mandatory to evaluate catalytic (labile) ferrous iron (catalytic Fe(II)) and to study iron manipulation in ovarian endometriotic lesions. By the use of tissues from women of ovarian endometriosis as well as endometrial tissue from women with and without endometriosis, we for the first time performed histological analysis and cellular detection of catalytic Fe(II) with a specific fluorescent probe (HMRhoNox-M), and further evaluated iron transport proteins in the human specimens and in co-culture experiments using immortalized human eutopic/ectopic endometrial stromal cells (ESCs) in the presence or absence of epithelial cells (EpCs). The amounts of catalytic Fe(II) were higher in ectopic endometrial stromal cells (ecESCs) than in normal eutopic endometrial stromal cells (n-euESCs) both in the tissues and in the corresponding immortalized ESCs. ecESCs exhibited higher transferrin receptor 1 expression both in vivo and in vitro and lower ferroportin expression in vivo than n-euESCs, leading to sustained iron uptake. In co-culture experiments of ESCs with iron-loaded EpCs, ecESCs received catalytic ferrous iron from EpCs, but n-euESCs did not. These data suggest that ecESC play a protective role for cancer-target epithelial cells by collecting excess iron, and that these characteristics are retained in the immortalized ecESCs.

Optimization of biguanide derivatives as selective antitumor agents blocking adaptive stress responses in the tumor microenvironment

Adaptive cellular responses resulting from multiple microenvironmental stresses, such as hypoxia and nutrient deprivation, are potential novel drug targets for cancer treatment. Accordingly, we focused on developing anticancer agents targeting the tumor microenvironment (TME). In this study, to search for selective antitumor agents blocking adaptive responses in the TME, thirteen new compounds, designed and synthesized on the basis of the arylmethylbiguanide scaffold of phenformin, were used in structure activity relationship studies of inhibition of hypoxia inducible factor (HIF)-1 and unfolded protein response (UPR) activation and of selective cytotoxicity under glucose-deprived stress conditions, using HT29 cells. We conducted luciferase reporter assays using stable cell lines expressing either an HIF-1-responsive reporter gene or a glucose-regulated protein 78 promoter-reporter gene, which were induced by hypoxia and glucose deprivation stress, respectively, to screen for TME-targeting antitumor drugs. The guanidine analog (compound 2), obtained by bioisosteric replacement of the biguanide group, had activities comparable with those of phenformin (compound 1). Introduction of various substituents on the phenyl ring significantly affected the activities. In particular, the o-methylphenyl analog compound 7 and the o-chlorophenyl analog compound 12 showed considerably more potent inhibitory effects on HIF-1 and UPR activation than did phenformin, and excellent selective cytotoxicity under glucose deprivation. These compounds, therefore, represent an improvement over phenformin. They also suppressed HIF-1- and UPR-related protein expression and secretion of vascular endothelial growth factor-A. Moreover, these compounds exhibited significant antiangiogenic effects in the chick chorioallantoic membrane assay. Our structural development studies of biguanide derivatives provided promising candidates for a novel anticancer agent targeting the TME for selective cancer therapy, to be subjected to further in vivo study.

Non-thermal plasma induces a stress response in mesothelioma cells resulting in increased endocytosis, lysosome biogenesis and autophagy

Abstract Non‐thermal plasma (NTP) is a potential new therapeutic modality for cancer. However, its mechanism of action remains unclear. Herein, we studied the effect of NTP on mesothelioma cells and fibroblasts to understand its anti‐proliferative efficacy. Interestingly, NTP demonstrated greater selective anti‐proliferative activity against mesothelioma cells relative to fibroblasts than cisplatin, which is used for mesothelioma treatment. The anti‐proliferative effect of NTP was enhanced by pre‐incubation with the cellular iron donor, ferric ammonium citrate (FAC), and inhibited by iron chelation using desferrioxamine (DFO). Three oxidative stress probes (CM‐H2DCFDA, MitoSOX and C11‐BODIPY) demonstrated reactive oxygen species (ROS) generation by NTP, which was inhibited by DFO. Moreover, NTP decreased transferrin receptor‐1 and increased ferritin‐H and ‐L chain expression that was correlated with decreased iron‐regulatory protein expression and RNA‐binding activity. This regulation was potentially due to increased intracellular iron in lysosomes, which was demonstrated via the Fe(II)‐selective probe, HMRhoNox‐M, and was consistent with autophagic‐induction. Immunofluorescence using LysoTracker and Pepstatin A probes demonstrated increased cellular lysosome content, which was confirmed by elevated LAMP1 expression. The enhanced lysosomal biogenesis after NTP could be due to the observed increase in fluid‐phase endocytosis and early endosome formation. These results suggest NTP acts as a stressor, which results in increased endocytosis, lysosome content and autophagy. In fact, NTP rapidly increased autophagosome formation, as judged by increased LC3B‐II expression, which co‐localized with LAMP1, indicating autophagolysosome formation. Autophagic‐induction by NTP was confirmed using electron microscopy. In summary, NTP acts as a cellular stressor to rapidly induce fluid‐phase endocytosis, lysosome biogenesis and autophagy. Graphical abstract Figure. No Caption available. HighlightsNon‐thermal plasma (NTP) is a potential new therapeutic modality for cancer.Malignant mesothelioma cells were more sensitive to NTP than fibroblasts.Anti‐proliferative effect of NTP was dependent on iron and oxidative stress.NTP significantly increased catalytic Fe(II) in mesothelioma cells.NTP increased fluid‐phase endocytosis, lysosome biogenesis and autophagy.