Transcriptome analysis of cultured human vascular endothelial cells after γ-ray irradiation and correlation analysis with ATP, ADP, and adenosine as secondary soluble factors.

Vascular endothelial cells serve as barriers between blood components and subendothelial tissue and regulate the blood coagulation-fibrinolytic system. Ionizing radiation is a common physical stimulant that induces a bystander effect whereby irradiated cells influence neighboring cells through signalings, including purinergic receptor signaling, activated by adenosine 5'-triphosphate (ATP), adenosine 5'-diphosphate (ADP), and adenosine as secondary soluble factors. Human vascular endothelial EA.hy926 cells were cultured and irradiated with γ-rays or treated with ATP, ADP, or adenosine under non-toxic conditions. RNA-seq, gene ontology, and hierarchical clustering analyses were performed. The transcriptome analysis of differentially expressed genes in vascular endothelial cells after γ-ray irradiations suggests that the change of gene expression by γ-irradiation is mediated by ATP and ADP. In addition, the expression and activity of the proteins related to blood coagulation and fibrinolysis systems appear to be secondarily regulated by ATP and ADP in vascular endothelial cells after exposure to γ-irradiation. Although it is unclear whether the changes of the gene expression related to blood coagulation and fibrinolysis systems by γ-irradiation affected the increased hemorrhagic tendency through the exposure to γ-irradiation or the negative feedback to the activated blood coagulation system, the present data indicate that toxicity associated with γ-irradiation involves the dysfunction of vascular endothelial cells related to the blood coagulation-fibrinolytic system, which is mediated by the signalings, including purinergic receptor signaling, activated by ATP and ADP.

Lead suppresses perlecan expression via EGFR-ERK1/2-COX-2-PGI2 pathway in cultured bovine vascular endothelial cells.

Vascular endothelial cell growth is essential for the repair of intimal injury. Perlecan, a large heparan sulfate proteoglycan, intensifies fibroblast growth factor-2 (FGF-2) signaling as a co-receptor for FGF-2 and its receptor, and promotes the proliferation of vascular endothelial cells. Previously, we reported that 2 µM of lead, a toxic heavy metal, downregulated perlecan core protein expression and then suppressed the growth of vascular endothelial cells. However, since the mechanisms involved in the repression of perlecan by lead remains unclear, we analyzed its detailed signaling pathway using cultured bovine aortic endothelial cells. Our findings indicate that 2 µM of lead inhibited protein tyrosine phosphatase (PTP) activity and induced cyclooxygenase-2 (COX-2) via phosphorylation of the epidermal growth factor receptor (EGFR) and its downstream extracellular signal-regulated kinases (ERK1/2). In addition, among the prostanoids regulated by COX-2, prostaglandin I2 (PGI2) specifically contributes to the downregulation of perlecan expression by lead. This study revealed an intracellular pathway-the EGFR-ERK1/2-COX-2-PGI2 pathway activated by inhibition of PTP by lead-as a pathway that downregulates endothelial perlecan synthesis. The pathway is suggested to serve as a mechanism for the repression of perlecan expression, which leads to a delay in cell proliferation by lead.

Long-Term Exposure to Cadmium Causes Hepatic Iron Deficiency through the Suppression of Iron-Transport-Related Gene Expression in the Proximal Duodenum.

Cadmium (Cd) is an environmental pollutant that damages various tissues. Cd may cause a depletion of iron stores and subsequently an iron deficiency state in the liver. However, the molecular mechanism of decreased iron accumulation in the liver induced by long-term exposure to Cd is unknown. In this study, we investigated the hepatic accumulation of iron and the proximal duodenal expression of the genes involved in iron transport using mice chronically exposed to Cd. Five-week-old female C57BL/6J mice were fed a diet containing 300 ppm Cd for 12, 15, 19 and 21 months. The iron concentration in the liver was markedly decreased by Cd. Among iron-transport-related genes in the proximal duodenum, the gene expression of HCP1 and Cybrd1 was significantly decreased by Cd. HCP1 is an influx transporter of heme iron. Cybrd1 is a reductase that allows non-heme iron to enter cells. The expression of iron-transport-related genes on the duodenal basolateral membrane side was hardly altered by Cd. These results suggest that long-term exposure to Cd suppresses the expression of HCP1 and Cybrd1 in the proximal duodenum, resulting in reduced iron absorption and iron accumulation in the liver.

Pathogenesis of selective damage of granule cell layer in cerebellum of rats exposed to methylmercury.

Granule cell-selective toxicity of methylmercury in the cerebellum is one of the main unresolved issues in the pathogenesis of Minamata disease. Rats were orally administered methylmercury chloride (10 mg/kg/day) for 5 consecutive days, and their brains were harvested on days 1, 7, 14, 21, or 28 after the last administration for histological examination of the cerebellum. It was found that methylmercury caused a marked degenerative change to the granule cell layers but not to the Purkinje cell layers. The generative change of the granule cell layer was due to cell death, including apoptosis, which occurred at day 21 and beyond after the methylmercury administration. Meanwhile, cytotoxic T-lymphocytes and macrophages had infiltrated the granule cell layer. Additionally, granule cells are shown to be a cell type susceptible to TNF-α. Taken together, these results suggest that methylmercury causes small-scale damage to granule cells, triggering the infiltration of cytotoxic T-lymphocytes and macrophages into the granule cell layer, which secrete tumor necrosis factor-α (TNF-α) to induce apoptosis in granule cells. This chain is established based on the susceptibility of granule cells to methylmercury, the ability of cytotoxic T lymphocytes and macrophages to synthesize and secrete TNF-α, and the sensitivity of granule cells to TNF-α and methylmercury. We propose to call the pathology of methylmercury-induced cerebellar damage the "inflammation hypothesis."

Systematic Review and Meta-Analysis of In Vitro Anti-Human Cancer Experiments Investigating the Use of 5-Aminolevulinic Acid (5-ALA) for Photodynamic Therapy.

5-Aminolevulinic acid (5-ALA) is an amino acid derivative and a precursor of protoporphyrin IX (PpIX). The photophysical feature of PpIX is clinically used in photodynamic diagnosis (PDD) and photodynamic therapy (PDT). These clinical applications are potentially based on in vitro cell culture experiments. Thus, conducting a systematic review and meta-analysis of in vitro 5-ALA PDT experiments is meaningful and may provide opportunities to consider future perspectives in this field. We conducted a systematic literature search in PubMed to summarize the in vitro 5-ALA PDT experiments and calculated the effectiveness of 5-ALA PDT for several cancer cell types. In total, 412 articles were identified, and 77 were extracted based on our inclusion criteria. The calculated effectiveness of 5-ALA PDT was statistically analyzed, which revealed a tendency of cancer-classification-dependent sensitivity to 5-ALA PDT, and stomach cancer was significantly more sensitive to 5-ALA PDT compared with cancers of different origins. Based on our analysis, we suggest a standardized in vitro experimental protocol for 5-ALA PDT.

Induction of metallothionein isoforms in cultured bovine aortic endothelial cells exposed to cadmium.

Metallothionein (MT) is an inducible protein with cytoprotective activity against heavy metals such as cadmium, zinc, and copper. MT-1 and MT-2 are the isoforms of MT induced by and bind the heavy metals. Bovine aortic endothelial cells contain three types of MT genes, namely, MT-1A, MT-1E, and MT-2A; however, the associated protein expression of these MT isoforms has not been identified. In the present study, the expression of MT subisoform proteins in cells treated with cadmium chloride was identified using a high-performance liquid chromatography-inductively coupled plasma-mass spectrometry system. It was revealed that: (1) transcriptional induction of MT-1A by cadmium was markedly more sensitive than that of MT-1E/2A; (2) MT-1A and MT-2A proteins were the predominant MT subisoforms induced by cadmium; and (3) there might be differentiation in the functions of MT-1 and MT-2 against cadmium cytotoxicity, although the actual roles of the MT isoforms in the cells were not distinct. This is the first study to show the differential induction of isoforms of MT proteins in vascular endothelial cells by cadmium.

Novel Photosensitizer ß-Mannose-Conjugated Chlorin e6 as a Potent Anticancer Agent for Human Glioblastoma U251 Cells.

A photosensitizer is a molecular drug for photodynamic diagnosis and photodynamic therapy (PDT) against cancer. Many studies have developed photosensitizers, but improvements in their cost, efficacy, and side effects are needed for better PDT of patients. In the present study, we developed a novel photosensitizer ß-mannose-conjugated chlorin e6 (ß-M-Ce6) and investigated its PDT effects in human glioblastoma U251 cells. U251 cells were incubated with ß-M-Ce6, followed by laser irradiation. Cell viability was determined using the Cell Counting Kit-8 assay. The PDT effects of ß-M-Ce6 were compared with those of talaporfin sodium (TS) and our previously reported photosensitizer ß-glucose-conjugated chlorin e6 (ß-G-Ce6). Cellular uptake of each photosensitizer and subcellular distribution were analyzed by fluorescence microscopy. ß-M-Ce6 showed 1000× more potent PDT effects than those of TS, and these were similar to those of ß-G-Ce6. ß-M-Ce6 accumulation in U251 cells was much faster than TS accumulation and distributed to several organelles such as the Golgi apparatus, mitochondria, and lysosomes. This rapid cellular uptake was inhibited by low temperature, which suggested that ß-M-Ce6 uptake uses biological machinery. ß-M-Ce6 showed potent PDT anti-cancer effects compared with clinically approved TS, which is a possible candidate as a next generation photosensitizer in cancer therapy.

Possible mechanism of heme oxygenase-1 expression in rat malignant meningioma KMY-J cells subjected to talaporfin sodium-mediated photodynamic therapy.

BACKGROUND: We previously demonstrated that heme oxygenase-1 (HO-1) induction may contribute to a protective response against photodynamic therapy (PDT) using talaporfin sodium (TS) in rat malignant meningioma KMY-J cells. In the present study, we examined the mechanism of HO-1 induction by PDT with TS (TS-PDT) in KMY-J cells. METHODS: KMY-J cells were incubated with 25 µM TS for 2 h and then exposed to 664 nm diode laser irradiation at 1 J/cm2. The gene and protein expression levels of HO-1 and hypoxia-inducible factor-1α (HIF-1α) were determined by real-time RT-PCR and western blot analysis, respectively. Cell viability was measured using the cell counting kit-8 assay. RESULTS: mRNA and protein levels of HO-1 in KMY-J cells were increased significantly at 3, 6, and 9 h after laser irradiation and the increased mRNA level of HO-1 was decreased by antioxidant N-acetyl cysteine treatment. The protein level of HIF-1α, which mediates transcriptional activation of the HO-1 gene, was increased significantly at 1 h after laser irradiation. Additionally, induction of mRNA expression of HO-1 by TS-PDT was diminished by HIF-1α inhibitor echinomycin. We also demonstrated that echinomycin significantly augmented the cytotoxic effect of TS-PDT. CONCLUSIONS: Our findings indicate that TS-PDT may induce HO-1 expression via reactive oxygen species production and then HIF-1 pathway activation in KMY-J cells, and the HO-1 induction may cause attenuation of the therapeutic effect of TS-PDT.

Cadmium induces iron deficiency anemia through the suppression of iron transport in the duodenum.

Cadmium (Cd) is an environmental contaminant that triggers toxic effects in various tissues such as the kidney, liver, and lung. Cd can also cause abnormal iron metabolism, leading to anemia. Iron homeostasis is regulated by intestinal absorption. However, whether Cd affects the iron absorption pathway is unclear. We aimed to elucidate the relationship between the intestinal iron transporter system and Cd-induced iron deficiency anemia. C57BL/6J female and male mice, 129/Sv female mice, and DBA/2 female mice were given a single oral dose of CdCl2 by gavage. After 3 or 24 h, Cd decreased serum iron concentrations and inhibited the expression of iron transport-related genes in the duodenum. In particular, Cd decreased the levels of divalent metal transporter 1 and ferroportin 1 in the duodenum. In addition, human colon carcinoma Caco-2 cells were treated with CdCl2. After 72 h, Cd decreased the expression of iron transport-related factors in Caco-2 cells with a pattern similar to that seen in the murine duodenum. These findings suggest that Cd inhibits iron absorption through direct suppression of iron transport in duodenal enterocytes and contributes to abnormal iron metabolism.

Synthesis and anticancer activity of bis(2-arylimidazo[1,2-a]pyridin-3-yl) selenides and diselenides: the copper-catalyzed tandem C-H selenation of 2-arylimidazo[1,2-a]pyridine with selenium.

Most heteroaryl selenides and diselenides are biologically active, with some reported to act as antioxidants and show activities that are medicinally relevant; hence, the development of efficient methods for their synthesis is an important objective. Herein, a simple method for the synthesis of selenides and diselenides bearing imidazo[1,2-a]pyridine rings and their anticancer activity are described. The double C-H selenation of imidazo[1,2-a]pyridine with Se powder was catalyzed by CuI (10 mol %) ligated with 1,10-phenanthroline (10 mol %) at 130 °C under aerobic conditions. The selenides or diselenides were prepared almost selectively using selenium powder in an appropriate quantity under otherwise identical reaction conditions. The prepared selenides and diselenides bearing two imidazo[1,2-a]pyridine rings were all novel compounds. Among the prepared diselenides and selenides that exhibited cytotoxicity against cancer cells, bis[2-(4-methoxyphenyl)imidazo[1,2-a]pyridin-3-yl] diselenide showed an excellent anticancer activity and low cytotoxicity toward noncancer cells, suggesting that this diselenide is a potential lead compound for anticancer therapy.