Tumor angiogenesis, the formation of new blood capillaries by vascular endothelial cells from existing vessels, is an important mechanism for supplying nutrients, oxygen, growth factors and others to tumor cells. Tumor cells trigger angiogenesis by secreting angiogenic factors, especially vascular endothelial growth factor (VEGF-A), 1) which plays an important role in the regulation of normal and abnormal angiogenesis.2) VEGF-A (commonly known as VEGF) was first reported as a vascular permeability-inducing factor secreted by tumor cells, and referred to as vascular permeability factor (VPF). Reactive oxygen species (ROS) are suggested to play an important role in angiogenesis. 10) Furthermore, there is increasing evidence of the involvement of H 2 O 2 in the regulation of angiogenesis.9,11-13) As well, a variety of cell lines derived from human tumors has been shown to produce large amounts of H 2 O 2 .14) Constitutive surveillance for cellular protection against oxidative stress is conferred by intracellular antioxidative agents.15) Excess amounts of ROS are toxic and cause a reduction of intracellular antioxidant levels.16) It has been reported that pretreatment of the heart with exogenous antioxidants improved its condition as a result of reducing ROS production.17) The VEGF-A gene is one that has its expression regulated by ROS, especially by H 2 O 2 . Additional data support that VEGF-A mRNA is up-regulated by H 2 O 2 in a dose-and time-dependent manner. 18,19) Taken together, these suggest that some endogenous as well as exogenous antioxidative agents can be used to regulate VEGF-A gene expression and/or H 2 O 2 production for therapeutic purposes.Electrolyzed reduced water (ERW) has attracted much attention because of its antioxidative potential. Water electrolysis typically produces two forms of water: reduced or alkaline (high pH) water near the cathode and an oxidized or acid (low pH) water near the anode. Applications of oxidized
We have established a cartilaginous fish cell line [Squalus acanthias embryo cell line (SAE)], a mesenchymal stem cell line derived from the embryo of an elasmobranch, the spiny dogfish shark S. acanthias. Elasmobranchs (sharks and rays) first appeared >400 million years ago, and existing species provide useful models for comparative vertebrate cell biology, physiology, and genomics. Comparative vertebrate genomics among evolutionarily distant organisms can provide sequence conservation information that facilitates identification of critical coding and noncoding regions. Although these genomic analyses are informative, experimental verification of functions of genomic sequences depends heavily on cell culture approaches. Using ESTs defining mRNAs derived from the SAE cell line, we identified lengthy and highly conserved gene-specific nucleotide sequences in the noncoding 3 UTRs of eight genes involved in the regulation of cell growth and proliferation. Conserved noncoding 3 mRNA regions detected by using the shark nucleotide sequences as a starting point were found in a range of other vertebrate orders, including bony fish, birds, amphibians, and mammals. Nucleotide identity of shark and human in these regions was remarkably well conserved. Our results indicate that highly conserved gene sequences dating from the appearance of jawed vertebrates and representing potential cis-regulatory elements can be identified through the use of cartilaginous fish as a baseline. Because the expression of genes in the SAE cell line was prerequisite for their identification, this cartilaginous fish culture system also provides a physiologically valid tool to test functional hypotheses on the role of these ancient conserved sequences in comparative cell biology.noncoding sequence ͉ sequence conservation ͉ Squalus acanthias E fforts toward a comprehensive compilation of cell biological, physiological, and genomic information from a variety of vertebrate species are providing a rich source of comparative data representing critical points in evolutionary divergence (1). Although available information is rapidly expanding for teleosts (bony fishes; ref.2), few cell biological or genomic data are available for the most primitive jawed vertebrates, the cartilaginous fish, class Chondrichthyes, largely represented by the order Elasmobranchii (3, 4). This information would extend comparative data through Ͼ450 million years of evolution. We have derived an embryonic mesenchymal stem cell line [Squalus acanthias embryo cell line (SAE)] from the elasmobranch S. acanthias, the spiny dogfish shark (5). This cell line from a cartilaginous fish provides a means to study the cell biology, physiology, and genomics of Chondrichthyes. Recently, the National Human Genome Research Institute targeted for comprehensive genomic sequencing Leucoraja erinacia, an elasmobranch of the superorder Squaliformes, related to S. acanthias, underscoring the value of Chondrichthyes for comparative biology (6) In efforts to explore the cell biology and genomics of c...
In the two-stage cell transformation theory, cancer cells first receive initiation, which is mainly caused by DNA damage, and then promotion, which enhances transformation. Murine Balb/c 3T3 cells are widely used for transformation experiments because they lose contact inhibition ability when transformed. Electrolyzed reduced water (ERW), which is produced near a cathode during electrolysis of water, is an alkaline drinking water that is beneficial to health. ERW contains a high concentration of dissolved hydrogen and scavenge reactive oxygen species (ROS), along with a small amount of platinum (Pt) nanoparticles (Pt nps) derived from Pt-coated titanium electrodes. Pt nps stably disperse in aqueous solution for a long time, and convert hydrogen molecules to active hydrogen (atomic hydrogen) that can scavenge ROS. Therefore, ERW supplemented with synthesized Pt nps is a model strong reduced water. This is the first report that ERW supplemented with synthesized Pt nps strongly prevents transformation of Balb/c 3T3 cells. ERW was prepared by electrolysis of 0.002 M NaOH solution using a batch-type electrolysis device. Balb/c 3T3 cells were treated with 3-methyl cholanthrene (MCA) as an initiation substance, followed by treatment with phorbol-12-myristate-13-acetate (PMA) as a promotion substance. MCA/PMA-induced formation of a transformation focus was strongly suppressed by ERW supplemented with Pt nps but not by ERW or Pt nps individually. ERW supplemented with Pt nps suppressed transformation at the promoter stage, not at initiation, suggesting that ERW supplemented with Pt nps suppressed the PMA-induced augmentation of intracellular ROS. ERW supplemented with Pt nps is a potential new antioxidant against carcinogenesis.Abbreviations: ASA-2P -ascorbic acid 2-phosphate; ERW -electrolyzed reduced water; FBS -fetal bovine serum; HEPES -2-[4-(2-hydroxyethyl)-1-piperazinyl]ethanesulfonic acid; MCA -3-methyl cholanthrene; MEM -minimum essential medium; PMA -phorbol-12-myristate-13-acetate; Pt nanoparticles -Pt nps; ROS -reactive oxygen species Cytotechnology (2005)
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