Yojiro Maehata

Acidic extracellular microenvironment and cancer

Acidic extracellular pH is a major feature of tumor tissue, extracellular acidification being primarily considered to be due to lactate secretion from anaerobic glycolysis. Clinicopathological evidence shows that transporters and pumps contribute to H+ secretion, such as the Na+/H+ exchanger, the H+-lactate co-transporter, monocarboxylate transporters, and the proton pump (H+-ATPase); these may also be associated with tumor metastasis. An acidic extracellular pH not only activates secreted lysosomal enzymes that have an optimal pH in the acidic range, but induces the expression of certain genes of pro-metastatic factors through an intracellular signaling cascade that is different from hypoxia. In addition to lactate, CO2 from the pentose phosphate pathway is an alternative source of acidity, showing that hypoxia and extracellular acidity are, while being independent from each other, deeply associated with the cellular microenvironment. In this article, the importance of an acidic extracellular pH as a microenvironmental factor participating in tumor progression is reviewed.

Effects of ascorbic acid and ascorbic acid 2-phosphate, a long-acting vitamin C derivative, on the proliferation and differentiation of human osteoblast-like cells

In order to investigate the effect of ascorbic acid (AsA) and ascorbic acid 2‐phosphate (Asc 2‐P), a long‐acting vitamin C derivative, on the growth and differentiation of human osteoblast‐like cells, we supplemented the culture medium of MG‐63 cells with various concentrations (0.25 to 1 mM) of these factors. Asc 2‐P significantly stimulated nascent cell growth at all concentrations in the presence of fetal bovine serum (FBS). On the other hand, AsA showed a growth repressive effect depending on its concentration, and that of FBS. Asc 2‐P also increased expression of osteoblast differentiation markers, such as collagen synthesis and alkaline phosphatase (ALP) activity. These stimulative activities of Asc 2‐P were attenuated by inhibitors of collagen synthesis, indicating that these effects were dependent on collagen synthesis. Electron micrographs of the cells showed the formation of a three‐dimensional tissue‐like structure endowed with a mature extracellular matrix in the presence of Asc 2‐P.

Direct Assessments of the Antioxidant Effects of Propofol Medium Chain Triglyceride/Long Chain Triglyceride on the Brain of Stroke-prone Spontaneously Hypertensive Rats Using Electron Spin Resonance Spectroscopy

Background:Antioxidant anesthetics such as propofol (2,6-diisopropylphenol) directly inhibit lipid peroxidation via the generation of reactive oxygen species. Currently, there are no other studies regarding the direct effects of propofol medium chain triglyceride/long chain triglyceride (MCT/LCT) on reactive oxygen species generation or in experimental models of reactive oxygen species–induced oxidative stress in the brain. Methods:The authors investigated the effects of propofol MCT/LCT on reactive oxygen species (hydroxyl radical or superoxide) by electron spin resonance spin trapping with 5,5-dimethyl-1-pyrroline-N-oxide. The effects of propofol MCT/LCT on oxidative stress in the brain of Wistar-Kyoto rats or stroke-prone spontaneously hypertensive rats were investigated by using an in vivo L-band electron spin resonance system to monitor the decay rate of 3-methoxycarbonyl-2,2,5,5-tetramethyl-pyrrolidine-1-oxyl as a nitroxyl spin probe. Results:These studies provided direct evidence that propofol MCT/LCT inhibited hydroxyl radical generation, but not superoxide generation. Regarding the hydroxyl radical from the Fenton system, it is likely to be due to the scavenging effects of vehicle. Anesthesia with propofol MCT/LCT reduced the degree of the high oxidative stress in the brain of stroke-prone spontaneously hypertensive rats. Conclusion:The current data show that propofol, mixed with clinical reagents (propofol MCT/LCT), resulted in the down-regulation of high oxidative stress due to scavenging hydroxyl radical, as demonstrated by in vitro or in vivo electron spin resonance analysis. These results led to reduced levels of hydroxyl radical, formed by brain injury such as stroke, and may therefore provide advantages for neuroprotection during anesthesia for craniotomy, e.g., in cases of brain disease.

Restoration of BRAK / CXCL14 gene expression by gefitinib is associated with antitumor efficacy of the drug in head and neck squamous cell carcinoma.

Clinical efficacy of gefitinib (ZD1839, Iressa), which is an inhibitor specific for epidermal growth factor (EGF) receptor tyrosine kinase, has been shown in non‐small‐cell lung carcinoma patients with EGF receptor mutations, so these mutations are useful marker(s) to find a responder for the drug. Recent studies have shown that the EGF receptor gene mutation is rare in squamous cell carcinoma in the esophageal and head and neck regions. We previously reported that the expression of the chemokine BRAK/CXCL14 in head and neck squamous cell carcinoma (HNSCC) cells was down‐regulated by EGF treatment, and that forced expression of BRAK in tumor cells decreased the tumorigenicity of the cells in xenografts. Thus, we investigated the relationship between restoration of BRAK expression by gefitinib and the efficacy of the drug for tumor suppression. We found that EGF down‐regulated BRAK expression through the MEK–extracellular signal regulated kinase pathway and that this down‐regulated expression was restored by gefitinib in vitro. Oral administration of gefitinib significantly (P < 0.001) reduced tumor growth of xenografts of three HNSCC cell lines (HSC‐2, HSC‐3, and HSC‐4), in female athymic nude mice, accompanied by an increase in BRAK expression specifically in tumor tissue. This tumor‐suppressing effect of the drug was not observed in the case of BRAK non‐expressing cells. Furthermore introduction of BRAK shRNA vector reduced both the expression levels of BRAK in HSC‐3 cells and the antitumor efficacy of gefitinib in vivo. Our data showing an inverse relationship between BRAK expression levels in tumor cells and the tumor growth rate indicate that the gefitinib‐induced increase in BRAK expression is beneficial for tumor suppression in vivo. (Cancer Sci 2009)

Reactive oxygen species production in mitochondria of human gingival fibroblast induced by blue light irradiation

In recent years, it has become well known that the production of reactive oxygen species (ROS) induced by blue-light irradiation causes adverse effects of photo-aging, such as age-related macular degeneration of the retina. Thus, orange-tinted glasses are used to protect the retina during dental treatment involving blue-light irradiation (e.g., dental resin restorations or tooth bleaching treatments). However, there are few studies examining the effects of blue-light irradiation on oral tissue. For the first time, we report that blue-light irradiation by quartz tungsten halogen lamp (QTH) or light-emitting diode (LED) decreased cell proliferation activity of human gingival fibroblasts (HGFs) in a time-dependent manner (<5 min). Additionally, in a morphological study, the cytotoxic effect was observed in the cell organelles, especially the mitochondria. Furthermore, ROS generation induced by the blue-light irradiation was detected in mitochondria of HGFs using fluorimetry. In all analyses, the cytotoxicity was significantly higher after LED irradiation compared with cytotoxicity after QTH irradiation. These results suggest that blue light irradiation, especially by LED light sources used in dental aesthetic treatment, might have adverse effects on human gingival tissue. Hence, this necessitates the development of new dental aesthetic treatment methods and/or techniques to protect HGFs from blue light irradiation during dental therapy.

Reactive oxygen species (ROS) reduce the expression of BRAK/CXCL14 in human head and neck squamous cell carcinoma cells.

Abstract The present study investigated the effects of oxidative stress induced by reactive oxygen species (ROS), such as hydrogen peroxide (H2O2) and hydroxyl radical (HO•), on the expression of both BRAK , which is also known as non-ELR motif angiostatic CXC chemokine ligand 14 (CXCL14), in head and neck squamous cell carcinoma (HNSCC) cells. When HNSCC cells were cultured in the presence of ROS, the expression of BRAK was significantly decreased whereas that of IL-8 was increased. Interestingly, the effects on the expression of both genes in HNSCC cells were much greater with HO▪ than with H2O2. The effects of ROS on both BRAK and IL-8 expression were attenuated by pre-treatment with N-acetyl-L-cysteine (NAC), epidermal growth factor receptor (EGFR), and mitogen-activated protein kinase (MAPK) inhibitors. These results indicate that oxidative stress induced by H2O2 or HO• stimulates angiogenesis and tumuor progression by altering the gene expression of BRAK and IL-8 via the EGFR/MEK/ERK pathway in human HNSCC cells.

Dental resin curing blue light induced oxidative stress with reactive oxygen species production

Dental resin curing blue light has been used in the treatment of tooth bleaching and to restore teeth with resin-based composite fillings. However, there has been little consideration of its effect on oral tissues such as dental pulp and oral mucosa. The aim of this study was to investigate whether dental resin curing blue light irradiation affects the dental pulp, especially the blood vessels that are known as the first target of reactive oxygen species (ROS), which play an important role in vascular reactivity. We found that blue light irradiation increased the level of lipid peroxidation in isolated rat aorta blood vessels by measuring malondialdehyde. Furthermore, cell proliferative activity was decreased in a time-dependent manner and apoptosis of human aorta vascular smooth muscle cells (VSMCs) was induced. These results indicated that (ROS) such as hydrogen peroxide and hydroxyl radicals were generated in VSMCs by irradiation with blue light, and they induced cytotoxicity associated with oxidative stress, which increased lipid peroxidation and apoptosis. In addition, N-acetyl-l-cysteine, which is a typical intracellular antioxidant, protected VSMCs against cytotoxicity associated with oxidative stress. These findings suggested that antioxidants may be used to prevent oxidative stress in dental pulp by repeated and/or multiple treatments with blue light irradiation in future dental treatments.

Reactive oxygen species generation in gingival fibroblasts of Down syndrome patients detected by electron spin resonance spectroscopy.

Abstract Oral manifestations of Down syndrome include high susceptibility to gingival inflammation with early onset and rapidly progressive periodontitis. The influence of reactive oxygen species (ROS) on periodontitis of Down syndrome is unclear. The aim of this study was to characterize ROS formation in Down syndrome-gingival fibroblasts (DS-GF) using electron spin resonance (ESR) spin trapping with 5,5-dimetyl-1-pyrolline-N-oxide (DMPO), and to determine whether ROS generation plays a role in the pathogenesis of periodontitis in Down syndrome patients. We observed formation of the DMPO-OH spin adduct, indicating HO• generation from cultured DS-GF and non-DS-GF. The increased HO• generation in cultured DS-GF was strongly decreased in the presence of the H2O2 scavenger, catalase, or the iron chelator, desferal. This may due to the enzymatic ability of over-expressed CuZn-superoxide dismutase in Down syndrome to catalyze the formation of H2O2 from O2•−, thereby increasing the availability of substrate H2O2 for the irondependent generation of HO• via the Fenton reaction, suggesting that HO• generated from DS-GF may be involved in progressive periodontitis of Down syndrome.

Suppressed rate of carcinogenesis and decreases in tumour volume and lung metastasis in CXCL14/BRAK transgenic mice

Cancer progression involves carcinogenesis, an increase in tumour size, and metastasis. Here, we investigated the effect of overexpressed CXC chemokine ligand 14 (CXCL14) on these processes by using CXCL14/BRAK (CXCL14) transgenic (Tg) mice. The rate of AOM/DSS-induced colorectal carcinogenesis in these mice was significantly lower compared with that for isogenic wild type C57BL/6 (Wt) mice. When tumour cells were injected into these mice, the size of the tumours that developed and the number of metastatic nodules in the lungs of the animals were always significantly lower in the Tg mice than in the Wt ones. Injection of anti-asialo-GM1 antibodies to the mice before and after injection of tumour cells attenuated the suppressing effects of CXCL14 on the tumor growth and metastasis, suggesting that NK cell activity played an important role during CXCL14-mediated suppression of tumour growth and metastasis. The importance of NK cells on the metastasis was also supported when CXCL14 was expressed in B16 melanoma cells. Further, the survival rates after tumour cell injection were significantly increased for the Tg mice. As these Tg mice showed no obvious abnormality, we propose that CXCL14 to be a promising molecular target for cancer suppression/prevention.

Calcium-calmodulin signaling induced by epithelial cell differentiation upregulates BRAK/CXCL14 expression via the binding of SP1 to the BRAK promoter region.

The chemokine BRAK/CXCL14 (BRAK) is expressed in normal squamous epithelium, but is not expressed or is expressed at negligible levels in head and neck squamous cell carcinoma. Malignant cells are known to be dedifferentiated compared with normal epithelial cells, suggesting a role for differentiation cues in the expression of BRAK. Thus, we examined the relationship between BRAK expression and stages of differentiation level in epithelial cells. Immunohistochemical analysis showed that BRAK protein was expressed in cells above the spinous cell layer in normal epithelia. In HSC-3 cells in culture, expression of BRAK mRNA was significantly upregulated by cell contact in a cell density-dependent manner, and mRNA expression of cell differentiation markers such as involucrin, cystatin-A, TGM1, TGM3, and TGM5 was concomitantly augmented. Furthermore, the upregulation of BRAK induced by cell contact was suppressed by chlorpromazine, a specific inhibitor of calmodulin. We previously reported that GC boxes and a TATA-like sequence in the BRAK promoter region are associated with the expression of BRAK. Using a promoter assay and ChIP, we demonstrated that binding of the stimulating protein-1 (SP1) transcription factor to a GC box upstream of the BRAK transcription start site was necessary for cell density-dependent upregulation of BRAK. These results indicated that upregulation of BRAK was accompanied by differentiation of epithelial cells induced by calcium/calmodulin signaling, and that SP1 binding to the BRAK promoter region played an important role in this signaling.