Radiation therapy is one of the choices to treat malignant tumors. Although radiation therapy has been established as an excellent local treatment for malignant tumors, existence of radiation ...resistant cell is a major problem to overcome. To reveal radioresistant mechanism, we investigated using clinically relevant radioresistant (CRR) cells that had been obtained by exposing to 2 Gy/day X-rays for more than 30 days. CRR cells show not only radioresistance but also resistant to anticancer drug docetaxel. CRR cells also show low frequency of DNA double strand breaks, low mitochondrial membrane potential and produce little amount of reactive oxygen species (ROS). However, the molecular mechanism to obtain radio-resistant is not clear. So, we try to reveal the resistant ability to other oxidative stress such as hydrogen peroxide (H2O2). First, we investigated the resistance of CRR cells against H2O2 using WST assay. As a result, these cells showed resistant to H2O2. Next, we examined gene expression and enzyme activity of catalase that is H2O2 catabolic enzyme. Catalase expression was up-regulated in CRR cells. However, catalase enzyme activity was down-regulated. We also investigated mitochondrial DNA (mtDNA) copy number. It was shown that the mtDNA copy number was decreased in CRR cells. In addition, the amount of ATP, ATPase gene expressions and plasma membrane potential were investigated in CRR cells. It was shown that the amount of ATP decreased, ATPase gene expressions were up regulated and plasma membrane potential were low in CRR cells. Furthermore, increase of internal H2O2 amount and lipid peroxidation was investigated. As a result, increase of internal H2O2 amount and lipid peroxidation were seen in parental cells 2h after H2O2 administration. On the other hand, increase of internal H2O2 amount and lipid peroxidation were not seen in CRR cells 2h after H2O2 administration. These results suggest that the decrease of cell response through plasma membrane component is the main factor rather than internal oxidative stress scavenging enzyme activity to obtain resistance against oxidative stress in CRR cells.
To achieve more effective cancer treatment, we have established and analyzed "clinically relevant radioresistant (CRR) cells" that can survive exposing to 2 Gy/day X-rays for more than 30 days. CRR ...cells show resistance against hydrogen peroxide (H2O2) that is one of the reactive oxygen species. However, the resistant mechanism to H2O2 has not been elucidated yet. Therefore, we investigated the involvement of iron ion in the resistant mechanism to H2O2 in CRR cells because iron ion has been reported to react with H2O2 and produce hydroxyl radical (･OH). ･OH have been shown to react with plasma membrane phospholipid and lead to cell death. Internal Fe2+ and ･OH amount were decreased in CRR cells compared with its parental cells. In addition, expression of ferritin, which is iron-binding protein, was increased in CRR cells. No internal H2O2 increase and no lipid peroxidation were seen in CRR cells after 50 µM H2O2 treatment for 2 hours, whereas internal H2O2 uptake and lipid peroxidation was increased after 50 µM H2O2 treatment for 2 hours in parental cells. Furthermore, Pretreatment of 10 µM of FeCl2 leads to more cell death after administration of 50 µM H2O2 in CRR cells. Administration of phospholipid also led to further cell death after 50 µM H2O2 treatment in CRR cells. These results suggest that intracellular Fe2+ content is very important against oxidative stress response in CRR cells and control of Fe2+ amount may be an effective option for cancer that is resistant to treatment.
An alkyl ammonium compound (cationic surface active agent), DDAC has been widely used as a detergent of germicides, antiseptics, and wood preservatives. The study was conduced in C57BL6/J mice ...following intratracheal instillation of DDAC to assess pulmonary toxic injuries. We initially intratracheally instilled to the lung with DDAC to determine a dose-related response. The high dose (0.1%) of DDAC treatment caused severe morbidity with pulmonary congestive edema. The middle dose (0.01%) of DDAC exposure increased inflammation, while the low dose (0.001%) did not, when examined inflammatory cells in bronchoalveolar lavages (BAL) from survivors 3 days after instillation. Next we examined time course changes in BAL and lung tissues from mice received the middle dose (0.01%) of DDAC instillation. The numbers of BAL macrophages, lymphocytes, and neutrophils were increases in a time-related manner, reaching peak at 7 days, in coincidence with increased IL-6, but not IL-10, -13, and IFN-γ. Glaucomatous fibrotic foci were strikingly observed in the lung at 3 days, and extended widely 7 days after instillation, with evidence of increased α-smooth muscle actin (α-SMA )and/or vimentin-positive cells. Developing fibrotic foci were likely associated with increased expression of TGF-β1 mRNA, but not TGF-β2 and -β3 mRNA. Phosphorylated smad2/3 was increased in fibrotic lungs, suggesting that there was activation of TGF-β signaling. To further explore the contribution of TGF-β/smad signaling, we isolated fibroblast-like cells from mouse lung, and then treated with a low dose (5μM) of DDAC. In consistent with in vivo data, TGF-β1 mRNA was increased by DDAC treatment in vitro, while TGF-β2 and -β3 mRNA were decreased. DDAC cancelled time-related reduction of α-SMA expression which was consistent with prolong phosphorylation of smad2/3. Pretreatment with SD208, a TGF-βRI (ALK5) kinase inhibitor attenuated DDAC-induced α-SMA expression through suppressing phosphorylation of smad2/3. These results suggested that DDAC instillation to mouse lung causes pulmonary fibrosis and these changes might be mediated by TGF-1/smad2/3 signaling.
Most wood preservatives developed to protect wood contain toxic or hazardous chemicals that can cause adverse impacts to human health. Amidst a rising tide of concern about the arsenical ...preservatives, especially Chromated Copper Arsenate, our recent research has been focused on understanding its toxic effects by oral and dermal exposure routes. Whereas chromium (Cr) and arsenic (As) within metal fumes impact respiratory diseases in human, the combined effects of these metals have not been elucidated. The present study was conduced in mice when intratracheally instilled As(V) and/or Cr(VI) to evaluate inflammatory response, cytotoxicity, and oxidative stress on respiratory system. C57BL/6 mice received 1250μg/kg of Cr(VI), 1560μg/kg of As(V), their mixture, or PBS. Bronchoalveolar lavage (BAL) and lungs were sampled 2 days after instillation. Instillation of As(V) or Cr(VI) increased the number of BAL neutrophils, which was greater in Cr(VI) exposure than in As(V). Concurrent As(V) exposure worsened Cr(VI)-induced BAL neutrophils, which were associated with increasing the numbers of BAL macrophages and lung neutrophils. These findings were supported by evidence of elevated IL-6, total protein, and LDH activity in BAL and caspase-3/7, -8 and -9 activities in the lung, along with enhanced ROS production and increased GSH and GSSG contents caused by co-treatment, when compared with the controls. These stress responses were possibly coordinated by MAPK signaling, as phosphorylation of ERK, SAPK/JNK and p38 MAPK was evident by combined treatment as well as Cr exposure. In vitro MTT assay revealed that Cr(VI) rather than As(V) induced cytotoxicity in a dose-dependent manner and co-treatment of As(V) enhanced Cr(VI)-induced cytotoxicity. These findings suggest that adverse effect of Cr(VI) is more evident than that of As(V) on acute respiratory system, and Cr(VI) exposure with As(V) makes its effect enhanced.
Lipopolysaccharide (LPS) produced by P. gingivalis has been reported as a major factor of periodontitis. Recently, LPS has been found to cause neuroinflammation, leading to neurodegeneration such as ...Alzheimer's disease. Neuroinflammation has also been shown to be involved in developmental disorders, but there are few reports on the relationships between LPS and developmental disorders. On the other hand, we have been shown that the repeated stress such as maternal separation causes behavioral abnormalities like developmental disorders via K＋-Cl- co-transporter 2 (KCC2) downregulation in mouse models. Therefore, we hypothesized that LPS causes neuroinflammation and decreases KCC2 that leads to developmental disorders. So, we analyzed KCC2 expression after LPS treatment using primary rat cerebral cortex cells and PC-12 cells. As a result, LPS treatment (10µg/ml) decreased KCC2 expression both in primary rat cerebral cortex cells and PC-12 cells. Gene expression of toll-like receptor4 (TLR4; LPS ligand), IL1-β (inflammation marker), and REST (KCC2 repressor) were up-regulated in PC-12 cells. Moreover, the expression of pWNK, which negatively regulates KCC2, was up-regulated by LPS treatment. These results indicate that we may propose the developmental disorder drug candidates by inhibiting this pathway in the brain.
To achieve more effective cancer treatment, we have established and analyzed "clinically relevant radioresistant (CRR) cells" that can survive exposing to 2 Gy/day X-rays for more than 30 days. CRR ...cells show resistance against hydrogen peroxide (H2O2) that is one of the reactive oxygen species. However, the resistant mechanism to H2O2 has not been elucidated yet. Therefore, we investigated the involvement of iron ion in the resistant mechanism to H2O2 in CRR cells because iron ion has been reported to react with H2O2 and produce hydroxyl radical (･OH). ･OH have been shown to react with plasma membrane phospholipid and lead to cell death. Internal Fe2+ and ･OH amount were decreased in CRR cells compared with its parental cells. In addition, expression of ferritin, which is iron-binding protein, was increased in CRR cells. No internal H2O2 increase and no lipid peroxidation were seen in CRR cells after 50 µM H2O2 treatment for 2 hours, whereas internal H2O2 uptake and lipid peroxidation was increased after 50 µM H2O2 treatment for 2 hours in parental cells. Furthermore, Pretreatment of 10 µM of FeCl2 leads to more cell death after administration of 50 µM H2O2 in CRR cells. Administration of phospholipid also led to further cell death after 50 µM H2O2 treatment in CRR cells. These results suggest that intracellular Fe2+ content is very important against oxidative stress response in CRR cells and control of Fe2+ amount may be an effective option for cancer that is resistant to treatment.
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