Ryuji Asano

Peroxisome proliferator-activated receptor δ as a molecular target to regulate lung cancer cell growth

It has been assumed that prostaglandin (PG)I2 signaling contributes to the negative growth control of lung cancer cells; however, the mechanism remains unresolved. PGI2 functions through a cell surface G protein‐coupled receptor (prostaglandin I2‐binding receptor, IP) and also exerts an effect by interacting with a nuclear hormone receptor, peroxisome proliferator‐activated receptor δ (PPARδ). We found that PPARδ was a key molecule of PGI2 signaling to give negative growth control of lung cancer cells (A549), using carbarprostacyclin, a PGI2 agonist for IP and PPARδ, and L‐165041, a PPARδ agonist. Furthermore, PPARδ‐induced cell growth control was reinforced by the inhibition of cyclooxygenase. These results suggest that PPARδ activation under the suppression of PG synthesis is important to regulate lung cancer cell growth.

Down-regulation of connexin 32 gene expression through DNA methylation in a human renal cell carcinoma cell.

Background: We have recently reported that connexin (Cx) 32 is down-regulated in a human renal cell carcinoma (RCC) cell (Caki-2 cell). Hypothesis: We postulated that the down-regulation of Cx32 gene in the RCC cell is due to hypermethylation of its promoter region. Methods: We estimated methylation status in the promoter region of Cx32 gene in the RCC cell by DNA digestion with methylation-sensitive restriction enzyme and PCR, and methylation-specific PCR (MSP). We also checked the recovery of Cx32 gene expression in the RCC cell treated with a DNA methyltranferase inhibitor, 5-Aza-2’-deoxycytidine (5-Aza-CdR). Results: Treatment with 5-Aza-CdR resulted in induction of Cx32 expression in the RCC cell. Hypermethylation of the Cx32 promoter region in the RCC cell was confirmed by DNA digestion with methylation-sensitive restriction enzyme and PCR, and MSP. Conclusion: We suggest that hypermethylation in the promoter region is a mechanism for the Cx32 gene repression in the RCC cell.

Contribution of the Src family of kinases to the appearance of malignant phenotypes in renal cancer cells

Although the constitute activation of the Src family of kinases (Src) has been established as a poor prognostic factor in several types of cancer, the role of Src in renal cell carcinoma (RCC) has not been defined. This study aimed to determine whether Src could contribute to the appearance of malignant phenotypes in RCC. The role of Src in the appearance of malignant phenotypes in RCC was examined in two human renal cancer cell lines, Caki‐1 from human metastatic RCC and ACHN from human primary RCC. Src activity in Caki‐1 cells was higher than that in ACHN cells, and this difference corresponded to the difference of PP1 (a Src family inhibitor)‐induced cytotoxicity on the two cells. The difference in cytotoxicity between the cells did not depend on cell cycle regulation but on the induction of apoptosis, and the difference in apoptosis particularly related to the reduction of the Bcl‐xL level. Furthermore, in Caki‐1 cells with higher Src activity, Src stimulated the production of vascular endothelial growth factor (VEGF), partially via the activation of Stat3, and the inhibition of Src activity caused a reduction of the VEGF level in serum, angiogenesis, and tumor development in a xenograft model. These results suggested that Src contributed to the appearance of malignant phenotypes in renal cancer cells, particularly due to the resistance against apoptosis by Bcl‐xL and angiogenesis stimulated by Src‐Stat3‐VEGF signaling.

The hyaluronan synthesis inhibitor 4-methylumbelliferone exhibits antitumor effects against mesenchymal-like canine mammary tumor cells

Hyaluronan (HA), a principal constituent of the extracellular matrix (ECM), mediates growth and metastasis of tumor cells. The role of HA in the epithelial-mesenchymal transition (EMT) is well known, and increased ECM remodeling is observed in mesenchymal-like cells. The HA synthesis inhibitor 4-methylumbelliferone (4-MU) is anti-tumorigenic for various malignant tumors. However, the antitumor effect of 4-MU against canine mammary tumor cells that possess a mesenchymal-like phenotype is unclear. We examined the antitumor effect of 4-MU on CF41.Mg mesenchymal-like canine mammary tumor cells. We investigated the influence of 4-MU on the expression of HA synthase (HAS) 1-3 mRNA and observed dose-dependent downregulation of HAS2 mRNA at 24-72 h; in contrast, HAS3 expression was elevated at 24 h. Thus, 4-MU inhibited HA synthesis via HAS2 repression. 4-MU also inhibited cell proliferation and induced apoptosis in the CF41.Mg cells. Our experiments showed that 4-MU-induced apoptosis in CF41.Mg cells involved induction of the pro-apoptotic gene BAX. We also assessed motility and found that 4-MU reduced chemokinesis and chemotaxis in CF41.Mg cells. Our data suggest that 4-MU may serve as a candidate therapeutic agent for the treatment of canine mammary tumors. Since 4-MU exhibits antitumor activity in mesenchymal-like cells, it may also be a useful inhibitor of canine mammary tumor invasion and metastasis.

Twenty-eight element concentrations in mane hair samples of adult riding horses determined by particle-induced X-ray emission

The concentrations of 28 elements (Al, Br, Ca, Cl, Co, Cu, Cr, Fe, Ga, Hg, K, Mg, Mn, Mo, Na, Nb, Ni, P, Pb, Rb, S, Se, Si, Sr, Ti, V, Y, and Zn) were measured in mane hair by the particle-induced X-ray emission method. Except for Br, Cl, K, S, and P, the trace element concentrations in mane hair of horses are similar to literature values for human hair. The values obtained are not dependent on the horses age, breed, and sex and could be used as reference values in the assessment of diseases and nutritional status in equines.

Usefulness of selective COX-2 inhibitors as therapeutic agents against canine mammary tumors

Cyclooxygenase-2 (COX-2) is a key enzyme for converting arachidonic acids to prostanoids, which are known to be induced during inflammation and cancer initiation. Previously, it has been reported that COX inhibitors, such as aspirin, reduce the incidence of human colorectal cancer; therefore, it is widely believed that COX-2 is a potential therapeutic and chemoprevention target for several types of human cancer. However, whether selective COX-2 inhibitors have antitumor effects against canine mammary tumor cells remains unclear. In the present study, to elucidate the antitumor effect of selective COX-2 inhibitors against canine mammary tumors, we investigated the antitumor effects of meloxicam, etodolac and celecoxib using COX-2-expressing canine mammary tumor (CF33) cells. We analyzed the effects of selective COX-2 inhibitors on COX-2 protein expression levels in CF33 cells. Celecoxib (100 µM) was found to induce downregulation of COX-2 protein expression. We examined the effect of selective COX-2 inhibitors on CF33 cell proliferation. All the selective COX-2 inhibitors suppressed CF33 cell growth. Specifically, etodolac and celecoxib inhibited cell proliferation via a decrease in S-phase cells and an increase in G0/G1 arrest. We examined the apoptotic effect of selective COX-2 inhibitors on CF33 cells. Our data suggested that etodolac and celecoxib induced apoptosis in CF33 cells. In particular, celecoxib led to apoptosis mediated by the activation of the mitochondrial apoptosis pathway, including the upregulation of BAX expression, downregulation of Bcl-2 expression and activation of caspase-3/7. Furthermore, celecoxib increased the percentages of cells in both early apoptosis and late apoptosis. Our results revealed that celecoxib induced apoptosis and cell cycle arrest in CF33 cells. The data suggested that celecoxib is the most viable candidate as a therapeutic agent for the treatment of canine mammary tumors. Furthermore, our findings provide the first indication that COX-2 inhibition can provide a new therapeutic strategy for treating canine mammary tumors.

4-methylumbelliferone leads to growth arrest and apoptosis in canine mammary tumor cells.

Hyaluronan (HA), a major component of the extracellular matrix (ECM), is synthesized by HA synthase (HAS) 1, HAS2 and HAS3 and is intricately involved in cell growth and metastasis. The HA synthesis inhibitor 4-methylumbelliferone (4-MU) has been reported to exhibit anticancer properties in various types of malignant tumors. However, the underlying mechanisms at the molecular and cellular levels remain unclear. In this study, to establish an animal model for studying the function of HA in human breast cancer, we investigated the antitumor effects of 4-MU using canine mammary tumor (CF33) cells. First, we investigated the effects of 4-MU on HA production in CF33 cells. Quantitative analysis of HA in culture media showed that 4-MU inhibited HA synthesis, accompanied by downregulation of HAS2 mRNA levels, in a dose-dependent manner at 24-72 h. Additionally, we observed a 4-MU-mediated decrease in the extent of the cell-associated HA matrix. We examined the effect of 4-MU on cell growth and apoptosis in CF33 cells. 4-MU markedly inhibited cell proliferation and induced apoptosis in CF33 cells. In particular, our experiments showed that the mechanism of 4-MU-induced apoptosis in CF33 cells involved increased levels of expression of pro-apoptotic BAX mRNA and protein molecules. These data suggest that 4-MU may be a candidate therapeutic agent for the treatment of canine mammary tumors. Furthermore, this study provides the first indication that the canine mammary tumor may be a suitable model for comparative study of the function of HA in human breast cancer.

α-Tocopheryloxybutyric acid enhances necrotic cell death in breast cancer cells treated with chemotherapy agent

The overexpression of HER-2 receptor contributes to malignant transformation of breast cancer cells. We have reported that alpha-tocopheryloxybutyric acid (TE), non-antioxidative vitamin E ether derivative inhibits the activation of HER-2 receptor. The present study was undertaken to estimate if TE could act as a useful anti-cancer agent against a breast cancer cell overexpressing HER-2 receptor (MDA-MB-453 cell line) in combination with a conventional chemotherapy agent, adriamycin (ADR). TE enhanced cytotoxic effect of ADR against the human breast cancer cell at low doses less than IC(50). The enhancing effect was mainly dependent on the elevation of necrotic-like cell death but not apoptotic cell death. In conjugation with this event, the inactivation of HER-2 receptor in the breast cancer cell was caused by the combination of TE with ADR. These results suggest that TE enhances necrotic-like cell death in the breast cancer cells and that the cell death relates to the inactivation of HER-2 receptor in the breast cancer cells.

Influence of the Coat Color on the Trace Elemental Status Measured by Particle-Induced X-ray Emission in Horse Hair

The influence of hair color on the trace elemental status in horses hair has been studied. A current analytical technique such as particle-induced X-ray emission (PIXE) used in this study has provided reliable, rapid, easy, and relatively inexpensive diagnostic methods. Twenty-eight elements (Al, Br, Ca, Cl, Co, Cu, Cr, Fe, Ga, Hg, K, Mg, Mn, Mo, Na, Nb, Ni, P, Pb, Rb, S, Se, Si, Sr, Ti, V, Y, and Zn) in mane hair were detected by the PIXE method. The gray hair contains significantly greter amounts of Cu, Ti, and Zn, and lower amounts of Br, Ca, Se, and Sr than those in other colored horse hairs (p<0.05). Those results measured in the horses hair were similar to those found in human and dog hair. When interpreting a result, it should be kept in mind that hair color, especially gray hair, influences the concentrations of some elements in horse hair.

Correlation between 25 element contents in mane hair in riding horses and atrioventricular block

The influence of atrioventricular block (AV-block) on the trace elemental status in a horse hair was studied. The particle-induced X-ray emission (PIXE) method has provided a reliable, rapid, easy, and relatively inexpensive diagnostic method. Twenty-five elements (Al, Br, Ca, Cl, Co, Cu, Cr, Fe, Hg, K, Mg, Mn, Na, Nb, Ni, P, Pb, Rb, S, Se, Si, Ti, Y, and Zn) in mane hair and serum were measured by the PIXE method. A horse hair with first- and second-degree AV-block contained significantly greater amounts of Br, Ca, Sr, and Zn than those of horses without electrocardiographic abnormalities, whereas there was no significant differences in the elemental contents of the serum of the both groups. Those results in contents of a horse hair suggest that the evaluation of the degree of ionic imbalance by this method might be used to predict the susceptibility of a horse to heart disease much before symptoms appear.