Shigeyuki Usui

Aminopeptidase N regulated by zinc in human prostate participates in tumor cell invasion

Aminopeptidase N (AP‐N) degrades collagen type IV and is proposed to play a role in tumor invasion. However, the precise functions of AP‐N in tumor cells and the relationship of AP‐N to prostate cancer remains unclear. In our study, we examined a possible role for zinc in the regulation of AP‐N enzymatic activity in relation to tumor cell invasion in human prostate. AP‐N purified from human prostate was irreversibly inhibited by low concentrations of zinc (Ki = 11.2 μM) and bestatin. AP‐N, which has zinc in the active center, was also inhibited by the chelating agents, EDTA, o‐phenanthroline and EGTA. EDTA was shown to remove zinc from the enzyme. When the effects of zinc and bestatin on invasion of PC‐3 cells were investigated in vitro using a Transwell cell‐culture chamber, zinc and bestatin effectively suppressed cell invasion into Matrigel at the concentration range of 50–100 μM. These results strongly suggest that the suppression of PC‐3 cell invasion by zinc is based on the inhibition of AP‐N activity by zinc. We also evaluated the expression of AP‐N to investigate the relationship with the progression of prostate disease in human cancerous prostate. AP‐N was found to be located at the cytoplasmic membranes of prostate gland epithelial cells and to be expressed more in prostate cancer, while the expression of prostate‐specific antigen (PSA), which is a useful marker for prostate cancer, was shown in normal and cancer tissues, suggesting that AP‐N is potentially a good histological marker of prostate cancer. Thus, highly expressed AP‐N in human cancerous prostate probably plays an important role in the invasion and metastasis of prostate cancer cells.

Tomato paste fraction inhibiting the formation of advanced glycation end-products.

A water-soluble and low-molecular-weight fraction (SB) was obtained from tomato paste. The effects of SB on the formation of advanced glycation end-products (AGE) in protein glycation were studied by the methods of specific fluorescence, ELISA and a Western blot analysis, using the anti-AGE antibody after incubating protein with sugar. The results suggest that SB had strong inhibitory activity, in comparison with aminoguanidine as a positive control, and that the inhibitory mechanism of SB differed from that of aminoguanidine to involve trapping of reactive dicarbonyl intermediates in the early stage of glycation. SB contained an antioxidant, rutin, which showed potent inhibitory activity. The results also suggest that rutin chiefly contributed to inhibiting the formation of AGE, and that other compounds in SB may also have been related to the activity.

Membrane trafficking of aquaporin 3 induced by epinephrine

We investigated the membrane trafficking of AQP3 induced by epinephrine in Caco-2 cells to clarify the digestive absorption of glycerol permeated by AQP3. Epinephrine was found to promote within 60 min the translocation of AQP3 from the cytoplasmic fraction to the plasma membrane. This increased trafficking of AQP3 was suppressed by phospholipase C and protein kinase C (PKC) inhibitors and a phorbol ester accelerated the trafficking of AQP3 to the membrane fraction. In contrast, adenylyl cyclase (AC) and protein kinase A (PKA) inhibitors did not have any effect on the increased in trafficking of AQP3 by epinephrine and an AC activator did not affect the trafficking of AQP3. Phosphorylation of a threonine (514) residue in PKC was detected upon the treatment with epinephrine and the temporal transitional pattern of this phosphorylation paralleled that of the increased trafficking of AQP3. These results suggest that PKC modulates the trafficking of AQP3.

Decreases of Metallothionein and Aminopeptidase N in Renal Cancer Tissues

Abstract Good molecular markers for investigating the biochemical differences between renal cancer and surrounding tissues have not yet been developed. Sixteen kidney samples (clear cell RCC) were investigated to determine the differences in the protein components between renal cancer and surrounding tissues, using HPLC analysis. The metallothionein (MT) and zinc levels were consistently lower in renal cancer tissues compared with in surrounding tissues. The mean concentration of MT in normal tissues surrounding renal tumors was about 15 times higher than that in cancer tissues. An immunohistochemical study confirmed that the expression of MT in renal cancer tissues was lower than that in adjacent normal tissues. The activities of aminopeptidases (APs) were significantly decreased in renal cancer tissues compared with in adjacent normal tissues. An immunohistochemical study and Western blot analysis confirmed that the expression of AP-N in renal cancer tissues was also lower than in adjacent normal tissues. These results suggest that the immunohistochemical detection of MT and AP-N could provide useful information as a pathological diagnostic tool for classifying renal cancer and surrounding tissues.

Transcriptional regulation of aquaporin 3 by insulin.

In the current study, we identified a regulatory factor for the transcription of aquaporin 3 (AQP3) whose expression is repressed by insulin. We constructed a luciferase reporter vector containing bp −1382 to −12 of the 5′‐flanking region of the AQP3 gene for a reporter gene assay and observed that luciferase activity in transfectants with the plasmid decreased on treatment with insulin. Serial deletion constructs revealed two regions responsible for the insulin‐mediated repression, one between bps −1382 and −780, and the other between bps −404 and −82. mRNA expression of forkhead box a2 (Foxa2), the binding site of which was located between bps −1382 and −780, was found to decrease on treatment with insulin. A mutant reporter plasmid with an altered Foxa2‐binding site and siRNA for the Foxa2 sequence counteracted the insulin‐mediated repression of AQP3 transcription. These results suggest that Foxa2 is one of the transcriptional regulators for AQP3 gene expression regulated by insulin. J. Cell. Biochem. 102: 1051–1058, 2007.

Imidazole-induced cell death, associated with intracellular acidification, caspase-3 activation, DFF-45 cleavage, but not oligonucleosomal DNA fragmentation

Intracellular acidification is known to be involved in the initiation phase of apoptosis. However, the necessity of intracellular acidic conditions in the execution phase of apoptosis remains unknown. In this study, we found that in HL-60 cells imidazole induces cell death, associated with intracellular acidification, caspase-3 activation and DFF-45 cleavage, but not oligonucleosomal DNA fragmentation. A caspase inhibitor prevented cell death but not intracellular acidification. When pHi was neutralized by changing from imidazole-containing medium to fresh medium, oligonucleosomal DNA fragmentation and increased caspase-3 activity was observed in the imidazole-treated HL-60 cells. Furthermore, the DNA fragmentation induced by intracellular neutralization was inhibited by caspase inhibitor treatment. These results indicate that imidazole induces caspase-dependent cell death, and suggest that maintaining pHi in the neutral range is essential for the induction of oligonucleosomal DNA fragmentation in the execution phase of apoptosis.

Involvement of interleukin 18 in indomethacin-induced lesions of the gastric mucosa in adjuvant-induced arthritis rat

It is well known that nonsteroidal anti-inflammatory drugs (NSAIDs) have significant side effects, such as gastroenteropathy, and rheumatoid arthritis patients taking NSAIDs are more susceptible to NSAIDs-induced gastric lesions in comparison with other patients. The pathogenic mechanism of these lesions is not fully understood. We demonstrate whether interleukin 18 (IL-18) expression relate the aggravation of gastric lesion in adjuvant-induced arthritis (AA) rats following the oral administration of indomethacin. Arthritis was induced by injecting 50 microl of a suspension of 10mg/ml heat-killed butyricum (Mycobacterium butyricum) in Bayol F oil into the plantar region of the right hind foot and tail of Dark Agouti rats resulting in an arthritis incidence of 100%. Two weeks after injection, the rats were administered indomethacin (40mg/kg) orally, and were killed under deep ether anesthesia 6h later. The gastric mucosa was then examined. Oral administration of indomethacin caused hemorrhagic lesions in the gastric mucosa of AA rats, and the lesion score for AA rats following indomethacin treatment was significantly higher than for normal rats administered indomethacin. The expression of the IL-18 mRNA and mature IL-18 protein in the gastric mucosa of AA rats administered indomethacin were also higher in comparison with normal rats receiving indomethacin. In addition, interferon-gamma and nitric oxide levels in the gastric mucosa of AA rats were increased by the oral administration of indomethacin. It is possible that IL-18 expression in AA rats is more sensitive to indomethacin, and the IL-18 may play a role in the aggravation of gastric lesions in AA rats treated with indomethacin.

Androgen receptor W741C and T877A mutations in AIDL cells, an androgen-independent subline of prostate cancer LNCaP cells

The androgen-independent LNCaP (AIDL) cell line was generated by maintaining prostate cancer LNCaP cells in a hormone-deprived medium. Notably, synthetic androgen R1881-related gene response is attenuated in AIDL cells as compared to the parental LNCaP cells. The aim of this study was to clarify the mechanisms underlying androgen sensitivity in AIDL cells. We first examined the expression of androgen receptor (AR) and its co-regulators. However, no significant difference in mRNA expression was found between LNCaP and AIDL cells. Remarkably, AR protein levels were induced by R1881 and DHT in LNCaP cells, but not in AIDL cells. We next performed the cDNA sequencing to detect mutations in the AR gene. The T877A mutation was detected both in LNCaP and AIDL cells. Furthermore, AIDL cells harbored a missense substitution (TGG → TGT) in the AR gene, which caused a point mutation at codon 741 (W741C). Double T877A and W741C AR mutants have been previously reported to exhibit reduced androgen sensitivity. Hence, the low-androgen-sensitive responses of AIDL cells may be explained, at least in part, by AR gene mutations.

AMP-activated protein kinase modulates the gene expression of aquaporin 9 via forkhead box a2

Aquaporin 9 (AQP9) is permeable to glycerol, which is a source material in lipogenesis and gluconeogenesis in the liver. We investigated the transcriptional regulation of the AQP9 gene by AMP-activated protein kinase (AMPK), known as an energy sensor in cells since AMPK contributes to the metabolism of carbohydrate, lipid, and protein by regulating the expression of many enzymes and transcription factors in metabolic pathways. An AMPK activator, 5-aminoimidazole-4-carboxamide-1-β-d-ribonucleoside (AICAR), was observed to suppress the expression of the AQP9 gene in HepG2 cells by promoting the phosphorylation of AMPK and AKT/PKB. Forkhead box a2 (Foxa2) was speculated to be one of the transcriptional regulators of AQP9 gene expression repressed by AICAR from the results of a reporter gene assay with a plasmid containing the promoter region of the AQP9 gene and knock-down of the Foxa2 gene by a specific siRNA. AICAR was determined to induce the phosphorylation and nuclear exclusion of Foxa2. Leptomycin B, inhibiting the binding of the nuclear exclusion signal sequence and chromosome region maintenance 1, prevented nuclear export of Foxa2 triggered by AICAR. These results suggest that the activated AMPK by AICAR causes suppression of the gene expression of AQP9 through transcriptional regulation by Foxa2.

Castration- and Aging-Induced Changes in the Expression of Zinc Transporter and Metallothionein in Rat Prostate

Prostate tissue contains high concentrations of zinc. Zinc content in the prostate gland changes in prostatic disease, such as benign prostate hyperplasia and prostate cancer, which occur more frequently with increasing age. Prostate zinc content is also known to decrease after castration in animal models. It is not clear how prostate zinc content is regulated; therefore, to clarify the mechanisms underlying zinc homeostasis, we examined zinc content and the expression of zinc transporters and metallothioneins in the prostates of aged or castrated rats. Zinc concentration was measured by flame atomic absorption spectrometry. The mRNA expression of zinc transporters and metallothioneins was determined by real-time reverse transcriptase polymerase chain reaction analysis. The expression of the zinc transporter Slc30a2 (Znt2) in ventral prostate (VP) of aged rats (21 months) was approximately 21-fold higher than that in VP of young rats (4 months), and zinc levels in VP of young rats increased significantly compared with that in aged rats. Zinc content in lateral prostate (LP) and dorsal prostate did not differ between young and aged rats. Decreased metallothionein-3 (Mt3) expression was observed in LP of castrated rats, and this reduction was prevented by testosterone replacement. Zinc content and Mt3 expression levels correlated significantly in rat LP. Our findings suggest that Mt3 could play a critical role in zinc homeostasis in rat LP.