Monascus pigment prevent the oxidative cytotoxicity in myotube derived hydrogen peroxide.

The amounts of Reactive oxygen species (ROS) become higher by strenuous exercises which consume larger amounts of oxygen in active muscles. Since these ROS directly injured muscles, the high ROS concentration involves muscle fatigue. Thus, an immediate ROS scavenging system in the muscle is desired. Since Monascus pigment (MP) involves physiologically active substances which scavenge ROS, it may be a clue to save the muscle injury. However, there are no reports examining MP effects on oxidative stress in skeletal muscle. In this study, we investigated the effect and mechanism of MP on skeletal muscle cells damaged by oxidative stress. The ability to directly eliminate ROS was evaluated by mixing MP solutions with •OH and O2 •-, a type of ROS. The effect of peroxidation in C2C12 cells was evaluated by cell viability assay and Western blotting. MP scavenges •OH and O2 •-. MP treatment increases the survival rate under oxidative stress. At that time, the expression of catalase was increased: the enzyme change H2O2 into H2O to rescue the cells under oxidative stress. We conclude that monascus pigment suppressed myotube damage under oxidative stress by both non-enzymatic ROS scavenging and up-regulation of catalase expression.

Reactive oxygen species induced by indomethacin enhance accumulation of heme carrier protein 1 and hematoporphyrin accumulation in vitro and in vivo in a brain tumor model.

Photodynamic therapy (PDT) is useful for various cancers such as high-grade glioma and cancers of other organs. However, the mechanism of tumor-specific accumulation of porphyrin is not clear. The authors previously reported that heme carrier protein 1 (HCP1) contributes to the transport of porphyrins; specifically, we showed that the production of cancer-specific reactive oxygen species from mitochondria (mitROS) leads in turn to enhanced HCP1 expression. Indomethacin (IND), a non-steroidal anti-inflammatory drug, increases ROS production by affecting mitochondrial electron transfer system. In the present work, the authors investigated the effect of pretreatment with IND on cancer-specific porphyrin accumulation, using both a glioma cell line and a rat brain tumor model. This work demonstrated that exposure of a rat glioma cell to IND results in increased generation of cancer-specific mitROS and accumulation of HCP1 expression and porphyrin concentration. Additionally, systemic dosing of a brain tumor animal model with IND resulted in elevated cellular accumulation of porphyrin in tumor cell. This is an effect not seen with normal brain tissue. Thus, the administration of IND increases intracellular porphyrin concentrations in tumor cell without exerting harmful effects on normal brain tissue, and increased porphyrin concentration in tumor cell may lead to improved PDT effect.

Enhancement of cytotoxic effects with ALA-PDT on treatment of radioresistant cancer cells.

Radiation therapy is a lower invasive local treatment than surgery and is selected as a primary treatment for solid tumors. However, when some cancer cells obtain radiotherapy tolerance, cytotoxicity of radiotherapy for cancer cells is attenuated. Photodynamic therapy (PDT) is a non-invasive cancer therapy combined with photosensitizers and laser irradiation with an appropriate wavelength. PDT is carried out for recurrent esophageal cancer patients after radiation chemotherapy and is an effective treatment for radiation-resistant tumors. However, it is not clear why PDT is effective against radioresistant cancers. In this study, we attempted to clear this mechanism using X-ray resistant cancer cells. X-ray resistant cells produce high amounts of mitochondria-derived ROS, which enhanced nuclear translocation of NF-κB, resulting in increased NO production. Moreover, the expression of PEPT1 that imports 5-aminolevulinic acid, the precursor of photosensitizers, was upregulated in X-ray resistant cancer cells. This was accompanied by an increase in intracellular 5-aminolevulinic acid-derived porphyrin accumulation, resulting in enhancement of PDT-induced cytotoxicity. Therefore, effective accumulation of photosensitizers induced by ROS and NO may achieve PDT after radiation therapy and PDT could be a promising treatment for radioresistant cancer cells.

Polphylipoprotein-induced autophagy mechanism with high performance in photodynamic therapy.

Polphylipoprotein (PLP) is a recently developed nanoparticle with high biocompatibility and tumor selectivity, and which has demonstrated unprecedentedly high performance photosensitizer in photodynamic therapy (PDT) and photodynamic diagnosis. On the basis of these discoveries, PLP is anticipated to have a very high potential for PDT. However, the mechanism by which PLP kills cancer cells effectively has not been sufficiently clarified. To comprehensively understand the PLP-induced PDT processes, we conduct multifaceted experiments using both normal cells and cancer cells originating from the same sources, namely, RGM1, a rat gastric epithelial cell line, and RGK1, a rat gastric mucosa-derived cancer-like mutant. We reveal that PLP enables highly effective cancer treatment through PDT by employing a unique mechanism that utilizes the process of autophagy. The dynamics of PLP-accumulated phagosomes immediately after light irradiation are found to be completely different between normal cells and cancer cells, and it becomes clear that this difference results in the manifestation of the characteristic effect of PDT when using PLP. Since PLP is originally developed as a drug delivery agent, this study also suggests the potential for intracellular drug delivery processes through PLP-induced autophagy.

Locally Administered Photodynamic Therapy for Cancer Using Nano-Adhesive Photosensitizer.

Photodynamic therapy (PDT) is a great potential anti-tumor therapy owing to its non-invasiveness and high spatiotemporal selectivity. However, systemically administered photosensitizers diffuse in the skin and the eyes for a long duration, which cause phototoxicity to bright light and sunlight. Therefore, following PDT, patients must avoid exposure of to light and sunlight to avoid this phototoxicity. In this study, we have developed a locally administered PDT using nano-adhesive porphyrin with polycations consisting of quaternary ammonium salt groups (aHP) as a photosensitizer. The aHP, approximately 3.0 nm in diameter, adhered the negatively charged cell membrane via electrostatic interaction. The aHP localized to the endosome via cell adhesion and induced apoptosis upon 635 nm light irradiation. On being administered subcutaneously on the tumor, 30% of the injected aHP remained in the administered sites. However, low-molecular-weight hematoporphyrin dihydrochloride (HP) disappeared due to rapid diffusion. PDT with locally administered aHP showed a higher anti-tumor effect after light irradiation at 635 nm for three days compared to low-molecular-weight HP. Intraperitoneal administration of HP caused severe phototoxicity upon irradiation with ultraviolet A at 10 J cm-2, whereas aHP did not cause phototoxicity because its diffusion into the skin could be suppressed, probably due to the high-molecular weight of aHP. Therefore, locally administered PDT with aHP is a potential PDT having high therapeutic efficacy without phototoxicity.

Therapeutic Potential of Seaweed-Derived Laminaran: Attenuation of Clinical Drug Cytotoxicity and Reactive Oxygen Species Scavenging.

ß-glucan has been shown to be effective for several diseases such as immune regulation and blood pressure suppression. Seaweed contains a ß-1,3/1,6-glucan called laminaran. The present commercial source of ß-glucan is black yeast; however, a fermentation process using organic carbon substrates makes production unsustainable, whereas macroalgae provide a sustainable alternative with the use of CO2 and seawater as growth substrates. However, bioactivity studies on laminaran are limited. We aimed to evaluate whether laminaran can scavenge reactive oxygen species (ROS) and attenuate cytotoxicity caused by clinical drugs such as indomethacin (Ind) and dabigatran (Dab). Electron spin resonance assay revealed that laminaran scavenged singlet oxygen (1O2) and superoxide anions (O2•-) directly but did not scavenge hydroxyl radicals (•OH). Mitochondrial ROS detection dye showed that laminaran scavenged mitochondrial O2•- produced upon administration of Ind or Dab. Moreover, significant reductions in •OH and peroxynitrate (ONOO-) levels were observed. Since •OH and ONOO- are generated from O2•- in the cells, laminaran could indirectly suppress the generation of •OH and ONOO- via the removal of O2•-. Both Ind and Dab induce cell injury via ROS production. Laminaran attenuated the cytotoxicity derived from these drugs and may represent a functional food with anti-aging and disease prevention properties.

Different nanobubbles mitigate cadmium toxicity and accumulation of rice (Oryza sativa L.) seedlings in hydroponic cultures.

Cadmium (Cd) contamination can pose a severe threat to food production and human health. The accumulation of Cd in rice will decrease rice biomass, photosynthetic activity, and antioxidant capacity, affecting crop yield. The effects of different nanobubbles on the growth and Cd accumulation of rice seedlings under hydroponic conditions were investigated in this study. The results showed that the biomass, photosynthetic pigment content, and antioxidant enzyme activity of rice seedlings decreased when treated with Cd alone and that Cd induced lipid peroxidation in rice seedlings. However, when different types of nanobubbles were introduced into the nutrient solution, the bioavailability of Cd in the solution was reduced. As a result, the Cd content in rice was significantly decreased compared to treatment with Cd alone. Nanobubbles increased the biomass of rice, enhanced photosynthesis, and improved the antioxidant capacity of rice by increasing antioxidant enzyme activities to alleviate Cd-induced oxidative stress. At the same time, nanobubbles increased the Fe content in rice, which decreased the Cd content, as Cd is antagonistic to Fe. In conclusion, these results suggested that nanobubbles are a potential method of mitigating Cd stress that may help to improve rice yield and could be further explored in production.

Cell-Level Analysis Visualizing Photodynamic Therapy with Porphylipoprotein and Talaporphyrin Sodium.

We revealed the difference in the mechanism of photodynamic therapy (PDT) between two photosensitizers: porphylipoprotein (PLP), which has recently attracted attention for its potential to be highly effective in treating cancer, and talaporphyrin sodium (NPe6). (1) NPe6 accumulates in lysosomes, whereas PLP is incorporated into phagosomes formed by PLP injection. (2) PDT causes NPe6 to generate reactive oxygen species, thereby producing actin filaments and stress fibers. In the case of PLP, however, reactive oxygen species generated by PDT remain in the phagosomes until the phagosomal membrane is destroyed, which delays the initiation of RhoA activation and RhoA*/ROCK generation. (4) After the disruption of the phagosomal membrane, however, the outflow of various reactive oxygen species accelerates the production of actin filaments and stress fibers, and blebbing occurs earlier than in the case of NPe6. (5) PLP increases the elastic modulus of cells without RhoA activity in the early stage. This is because phagosomes are involved in polymerizing actin filaments and pseudopodia formation. Considering the high selectivity and uptake of PLP into cancer cells, a larger effect with PDT can be expected by skillfully combining the newly discovered characteristics, such as the appearance of a strong effect at an early stage.

Acetic acid enhances the effect of photodynamic therapy in gastric cancer cells via the production of reactive oxygen species.

Acetic acid is a major component of vinegar and is reported to have beneficial health effects. Notably, it causes oxidative stress and enhances the production of reactive oxygen species (ROS) in gastric cancer cells. ROS play important roles in cellular signal transduction, resulting in the regulation of protein expression and apoptosis. We previously reported that ROS upregulate heme carrier protein 1 (HCP1). Moreover, ROS increase the cellular uptake of porphyrins, which are precursors of heme and substrates for uptake by HCP1. Therefore, we hypothesized that photodynamic therapy (PDT) for cancer treatment using laser irradiation and photosensitizers, such as porphyrin, is enhanced via ROS produced by acetic acid. Herein, we used the rat gastric mucosal cells, RGM1, its cancer-like mutated cells, RGK1, and a manganese superoxide dismutase (MnSOD)-overexpressing RGK cell line, RGK-MnSOD. We confirmed that cancer-specific cellular uptake of porphyrin is increased upon acetic acid treatment and enhances the PDT cytotoxicity in RGK-1, not in RGM-1 and RGK-MnSOD. We believe that this occurs because of the overproduction of ROS and subsequent upregulation of HCP1 in cancerous cells. In conclusion, acetic acid can elevate the effect of PDT by inducing cancer-specific HCP1 expression via ROS production.

Direct analysis of the actin-filament formation effect in photodynamic therapy.

Photodynamic therapy (PDT) is a method in which a photosensitizer is administered in vivo and irradiated with light to generate reactive oxygen species (ROS), thereby causing the selective death of cancer cells. Since PDT is a noninvasive cancer treatment method with few adverse effects, it has attracted considerable attention and is increasingly used. In PDT, there are two dominant processes based on the actin filament (A-filament) formation effect: the destruction of cells by necrosis and vascular shutdown. Despite the importance of its fine control, the mechanism of the reaction process from the generation of reactive oxygen by photoinduction inducing the formation of A-filament and its polymerization to form stress fibers (S-fibers) has not yet been clarified because, for example, it has been difficult to directly observe and quantify such processes in living cells by conventional methods. Here, we have combined atomic force microscopy (AFM) with other techniques to reveal the mechanism of the A-filament and S-fiber formation processes that underlie the cell death process due to PDT. First, it was confirmed that activation of the small G protein RhoA, which is a signal that induces an increase in A-filament production, begins immediately after PDT treatment. The production of A-filament did not increase with increasing light intensity when the amount of light was large. Namely, the activation of RhoA reached an equilibrium state in about 1 min: however, the production of A-filament and its polymerization continued. The observed process corresponds well with the change in the amount of phosphorylated myosin-light chains, which induce A-filament polymerization. The increase in the elastic modulus of cells following the formation of S-fiber was confirmed by AFM for the first time. The distribution of generated A-filament and S-fiber was consistent with the photosensitizer distribution. PDT increases A-filament production, and when the ROS concentration is high, blebbing occurs and cells die, but when it is low, cell death does not occur and S-fiber is formed. That is, it is expected that vascular shutdown can be controlled efficiently by adjusting the amount of photosensitizer and the light intensity.

Near-Infrared Light Irradiation of Porphyrin-Modified Gold Nanoparticles Promotes Cancer-Cell-Specific Cytotoxicity.

The use of nanoparticles has been investigated as a new cancer treatment. These can induce specific cytotoxicity in cancer cells. In particular, Au nanoparticles (AuNPs) have unique characteristics. The maximum absorption spectrum of AuNPs can be adjusted to modify their size or shape to absorb near-infrared light that can penetrate into tissue without photodamage. Thus, the combination of AuNPs and near-infrared light can be used to treat cancer in deep-seated organs. To obtain effective cancer-specific accumulation of AuNPs, we focused on porphyrin and synthesized a porphyrin-attached Au compound: Au-HpD. In this study, we investigated whether Au-HpD possesses cancer-specific accumulation and cytotoxicity. Intracellular Au-HpD accumulation was higher in cancer cells than in normal cells. In order to analyze the cytotoxicity induced by Au-HpD, cancer cells and normal cells were co-cultured in the presence of Au-HpD; then, they were subjected to 870 nm laser irradiation. We observed that, after laser irradiation, cancer cells showed significant morphological changes, such as chromatin condensation and nuclear fragmentation indicative of cell apoptosis. This strong effect was not observed when normal cells were irradiated. Moreover, cancer cells underwent cell apoptosis with combination therapy.

Stability and Free Radical Production for CO2 and H2 in Air Nanobubbles in Ethanol Aqueous Solution.

In this study, 8% hydrogen (H2) in argon (Ar) and carbon dioxide (CO2) gas nanobubbles was produced at 10, 30, and 50 vol.% of ethanol aqueous solution by the high-speed agitation method with gas. They became stable for a long period (for instance, 20 days), having a high negative zeta potential (-40 to -50 mV) at alkaline near pH 9, especially for 10 vol.% of ethanol aqueous solution. The extended Derjaguin, Landau, Verwey, and Overbeek (DLVO) theory was used to evaluate the nanobubble stability. When the nanobubble in ethanol alkaline aqueous solution changed to an acidic pH of around 5, the zeta potential of nanobubbles was almost zero and the decrease in the number of nanobubbles was identified by the particle trajectory method (Nano site). The collapsed nanobubbles at zero charge were detected thanks to the presence of few free radicals using G-CYPMPO spin trap reagent in electron spin resonance (ESR) spectroscopy. The free radicals produced were superoxide anions at collapsed 8%H2 in Ar nanobubbles and hydroxyl radicals at collapsed CO2 nanobubbles. On the other hand, the collapse of mixed CO2 and H2 in Ar nanobubble showed no free radicals. The possible presence of long-term stable nanobubbles and the absence of free radicals for mixed H2 and CO2 nanobubble would be useful to understand the beverage quality.

The Cytotoxicity of Doxorubicin Can Be Accelerated by a Combination of Hyperthermia and 5-Aminolevulinic Acid.

Chemotherapy is cytotoxic to various cancer cells and as well as normal cells. Thus, treatments that demonstrate selective cytotoxicity for cancer cells are desired. The combination of chemotherapy and other cancer therapies can show synergic cytotoxicity, which may be a clue to the nature of the involved cancer cellar-specific damage. We previously reported a phenomenon whereby mitochondrial reactive oxygen species (mitROS) regulate the expression transporters involved in anticancer drug transport and mitROS production is increased by hyperthermia. Moreover, the uptake of 5-aminolevulinic acid (ALA) was enhanced by the increase in mitROS production. In this study, we investigated whether the combination of hyperthermia and ALA can enhance the cytotoxicity of doxorubicin. MitROS production and ALA-derived porphyrin accumulation by hyperthermia (HT) were increased in a murine breast cancer cell line. The expression of solute carrier 15A1 (SLC15A1) upregulated and an ATP-binding cassette subfamily G member 2 (ABCG2) downregulated by HT. Since SLC15A1 is an accumulating transporter for ALA, while ABCG2 is a porphyrin efflux transporter, porphyrin accumulation was enhanced. ABCG2 is also a doxorubicin efflux transporter. Thus, ALA treatment accelerates the intracellular concentration of porphyrin, which acts as a competitive inhibitor of doxorubicin. Indeed, the amount of intracellular doxorubicin was increased by a combination of HT and ALA. The cytotoxicity of doxorubicin was also enhanced. This enhancement was observed in the human breast cancer cell line while it was not seen in normal cells. The combination of HT and ALA treatment can enhance the cancer-specific cytotoxicity of doxorubicin.

Dabigatran Etexilate Induces Cytotoxicity in Rat Gastric Epithelial Cell Line via Mitochondrial Reactive Oxygen Species Production.

Dabigatran is a novel oral anticoagulant that directly inhibits free and fibrin-bound thrombins and exerts rapid and predictable anticoagulant effects. While the use of this reagent has been associated with an increased risk of gastrointestinal bleeding, the reason why dabigatran use increases gastrointestinal bleeding risk remains unknown. We investigated the cytotoxicity of dabigatran etexilate and tartaric acid, the two primary components of dabigatran. The cytotoxicity of dabigatran etexilate and tartaric acid was measured in a cell viability assay. Intracellular mitochondrial reactive oxygen species (mitROS) production and lipid peroxidation were measured using fluorescence dyes. Cell membrane viscosity was measured using atomic force microscopy. The potential of ascorbic acid as an inhibitor of dabigatran cytotoxicity was also evaluated. The cytotoxicity of dabigatran etexilate was higher than that of tartaric acid. Dabigatran etexilate induced mitROS production and lipid peroxidation and altered the cell membrane viscosity. Ascorbic acid inhibited the cytotoxicity and mitROS production induced by dabigatran etexilate. Therefore, we attributed the cytotoxicity of dabigatran to dabigatran etexilate, and proposed that the cytotoxic effects of dabigatran etexilate are mediated via mitROS production. Additionally, we demonstrated that dabigatran cytotoxicity can be prevented via antioxidant treatment.

The cytotoxicity of cyclophosphamide is enhanced in combination with monascus pigment.

Monascus pigment is derived from red-mold rice fermented by monascus purpureus and utilized as a natural coloring agent and natural food additive in East Asia. Monascus pigment works as a radical scavenger. Some antioxidant combine cancer chemo-therapy to protect normal tissue because chemotherapy induce side effect for normal tissue. This combination therapy can attenuate the cytotoxicity of anti-cancer drugs by antioxidants effects. However, the effect of this combination therapy for cancer cells dose not investigate enough. In this study, we investigated the combination effect of anti-oxidants and anti-cancer drugs. We selected an anti-oxidant as monascus pigment and following four anti-cancer drugs: doxorubicin, tamoxifen, paclitaxicel, and cyclophosphamide. Combination treatment with monascus pigment and cyclophosphamide enhanced the cytotoxicity of cyclophosphamide. Moreover, this combination treatment accelerated apoptosis. The spot on TLC assay board of the monascus pigment and cyclophosphamide mixture is different from the spot of monascus pigment alone and cyclophosphamide alone. The interaction between monascus pigment and cyclo-phosphamide can produce some cytotoxicity compounds or accelerate intracellular cyclophosphamide accumulation. Hence, we concluded that the interaction of both cyclophosphamide and monascus pigment involved enhancement of cyclophosphamide cytotoxicity.

Porphylipoprotein Accumulation and Porphylipoprotein Photodynamic Therapy Effects Involving Cancer Cell-Specific Cytotoxicity.

In photodynamic therapy (PDT) for neoplasms, photosensitizers selectively accumulate in cancer tissue. Upon excitation with light of an optimal wavelength, the photosensitizer and surrounding molecules generate reactive oxygen species, resulting in cancer cell-specific cytotoxicity. Porphylipoprotein (PLP) has a porphyrin-based nanostructure. The porphyrin moiety of PLP is quenched because of its structure. When PLP is disrupted, the stacked porphyrins are separated into single molecules and act as photosensitizers. Unless PLP is disrupted, there is no photosensitive disorder in normal tissues. PLP can attenuate the photosensitive disorder compared with other photosensitizers and is ideal for use as a photosensitizer. However, the efficacy of PLP has not yet been evaluated. In this study, the mechanism of cancer cell-specific accumulation of PLP and its cytotoxic effect on cholangiocarcinoma cells were evaluated. The effects were investigated on normal and cancer-like mutant cells. The cytotoxicity effect of PLP PDT in cancer cells was significantly stronger than in normal cells. In addition, reactive oxygen species regulated intracellular PLP accumulation. The cytotoxic effects were also investigated using a cholangiocarcinoma cell line. The cytotoxicity of PLP PDT was significantly higher than that of laserphyrin-based PDT, a conventional type of PDT. PLP PDT could also inhibit tumor growth in vivo.

Free radical degradation in aqueous solution by blowing hydrogen and carbon dioxide nanobubbles.

The main findings are the hydroxyl radical scavenging and the superoxide anion diminishing by mixing the carbon dioxide (CO2) nanobubbles after hydrogen nanobubble blowing in water and alcohol aqueous solution. The nanobubbles produce the hydroxyl radical by ultrasonic waves, changing the pH and catalyst and so on, while the nanobubble is very reactive to scavenge free radicals. In this research especially hydrogen (4% H2 in argon) and CO2 nanobubbles have been blown into hydrogen peroxide (H2O2) added pure water, ethanol, and ethylene glycol aqueous solution through a porous ceramic sparger from the gas cylinder. The aqueous solutions with H2O2 are irradiated by ultraviolet (UV) light and the produced hydroxyl radical amount is measured with spin trapping reagent and electron spin resonance (ESR). The CO2 nanobubble blowing extremely has reduced the hydroxyl radical in water, ethanol, and ethylene glycol aqueous solution. On the other hand, when H2 nanobubbles are brown after CO2 nanobubble blowing, the hydroxyl radical amount has increased. For the disinfection test, the increase of hydroxyl radicals is useful to reduce the bacteria by the observation in the agar medium. Next, when the superoxide anion solution is mixed with nanobubble containing water, ethanol, and ethylene glycol aqueous solution, H2 nanobubble has reduced the superoxide anion slightly. The water containing both CO2 and H2 nanobubble reduces the superoxide anion. The less than 20% ethanol and the 30% ethylene glycol aqueous solution containing CO2 nanobubbles generated after H2 nanobubble blowing can diminish the superoxide anion much more. While the H2 nanobubble blowing after CO2 nanobubble blowing scavenges the superoxide anion slightly. The experimental results have been considered using a chemical reaction formula.

Mitochondrial reactive oxygen species and heme, non-heme iron metabolism.

Mitochondria are one of the most important organelles for eukaryotes, including humans, to produce energy. In the energy-producing process, mitochondria constantly generate reactive oxygen species as a by-product of electrons leaking out from the electron transport chain react with oxygen. The active oxygen, in turn, plays pivotal roles in mediating several signalings, including those that are implicated in the development of some diseases such as neurodegenerative disease, cardiovascular disease, and carcinogenesis. This signaling, derived from mitochondrial reactive oxygen species, also affects intracellular iron homeostasis by regulating the expression of transporters. Heme iron is incorporated into cells through HCP1, and non-heme iron is transported by DMT1 in absorptive cells. Intracellular iron is exported by ferroportin and bound with transferrin. In most types of cell including erythrocyte, transferrin-bound iron is incorporated through transferrin-transferrin receptor system. We previously reported that the expression of HCP1 and DMT1 was upregulated in cancer cells and that overexpression of manganese superoxide dismutase, which is a mitochondrial-specific superoxide dismutase, downregulated the expression. These findings indicate that mitochondrial reactive oxygen species is associated with iron-related oxidative reactions. Recently, a mitochondria-specific iron transporter, mitoferrin, was identified, and the relationships among mitochondria, iron transportation, and diseases have been increasingly clarified.

Elevated Production of Mitochondrial Reactive Oxygen Species via Hyperthermia Enhanced Cytotoxic Effect of Doxorubicin in Human Breast Cancer Cell Lines MDA-MB-453 and MCF-7.

Hyperthermia (HT) treatment is a noninvasive cancer therapy, often used with radiation therapy and chemotherapy. Compared with 37 °C, 42 °C is mild heat stress for cells and produces reactive oxygen species (ROS) from mitochondria. To involve subsequent intracellular accumulation of DOX, we have previously reported that the expression of ATP-binding cassette sub-family G member 2 (ABCG2), an exporter of doxorubicin (DOX), was suppressed by a larger amount of intracellular mitochondrial ROS. We then hypothesized that the additive effect of HT and chemotherapy would be induced by the downregulation of ABCG2 expression via intracellular ROS increase. We used human breast cancer cell lines, MCF-7 and MDA-MB-453, incubated at 37 °C or 42 °C for 1 h to clarify this hypothesis. Intracellular ROS production after HT was detected via electron spin resonance (ESR), and DOX cytotoxicity was calculated. Additionally, ABCG2 expression in whole cells was analyzed using Western blotting. We confirmed that the ESR signal peak with HT became higher than that without HT, indicating that the intracellular ROS level was increased by HT. ABCG2 expression was downregulated by HT, and cells were injured after DOX treatment. DOX cytotoxicity enhancement with HT was considered a result of ABCG2 expression downregulation via the increase of ROS production. HT increased intracellular ROS production and downregulated ABCG2 protein expression, leading to cell damage enhancement via DOX.

Virtual-freezing fluorescence imaging flow cytometry.

By virtue of the combined merits of flow cytometry and fluorescence microscopy, imaging flow cytometry (IFC) has become an established tool for cell analysis in diverse biomedical fields such as cancer biology, microbiology, immunology, hematology, and stem cell biology. However, the performance and utility of IFC are severely limited by the fundamental trade-off between throughput, sensitivity, and spatial resolution. Here we present an optomechanical imaging method that overcomes the trade-off by virtually freezing the motion of flowing cells on the image sensor to effectively achieve 1000 times longer exposure time for microscopy-grade fluorescence image acquisition. Consequently, it enables high-throughput IFC of single cells at >10,000 cells s-1 without sacrificing sensitivity and spatial resolution. The availability of numerous information-rich fluorescence cell images allows high-dimensional statistical analysis and accurate classification with deep learning, as evidenced by our demonstration of unique applications in hematology and microbiology.