Shunichiro Kubota

Formation of hydroxy radicals by environmental estrogen-like chemicals in rat striatum

We investigated effects of environmental estrogen-like chemicals, para-nonylphenol and bisphenol A, on hydroxy radical formation in the striatum of adult rats, using an in vivo microdialysis system. Para-nonylphenol significantly stimulated hydroxy radical formation in the striatum. Bisphenol A also increased hydroxy radical formation, albeit effect being slight. The formation of hydroxy radicals induced by para-nonylphenol was dose-dependently inhibited by tamoxifen, which suggests that the effect of this chemical was an estrogenic action via estrogen receptors. The results of the present study are the first demonstration on hydroxy radical formation induced by environmental estrogen-like chemicals and suggest that the in vivo microdialysis may be useful for evaluating toxic effects of environmental chemicals on nervous tissues.

Anti‐α3 integrin antibody induces the activated form of matrix metalloprotease‐2 (MMP‐2) with concomitant stimulation of invasion through matrigel by human rhabdomyosarcoma cells

Proteolytic enzymes, such as matrix metalloproteases (MMPs), play a pivotal role in cancer invasion and metastasis. Invasive human rhabdomyosarcoma cells (RD) secreted proMMP‐2 (72‐kDa progelatinase). We found that anti‐α3 and ‐α2 integrin antibodies induced the activated form of MMP‐2 and enhanced proMMP‐2 secretion by RD cells. The effect of anti‐α2 integrin antibody was less prominent than that seen with anti‐α3 integrin antibody. Moreover, we have found that anti‐α3 and ‐α2 integrin antibodies enhanced RD‐cell invasion through matrigel (reconstituted basement membrane) by 2.6‐and 2.0‐fold respectively this process was abrogated by neutralizing antibody to MMP‐2. These data suggest that signaling events induced by anti‐α3 integrin antibody may be involved in RD‐cell invasion as a result of modulation of matrix‐metalloprotease expression.

Molecular Cloning and Expression of Rabbit Sterol 12α-Hydroxylase

Sterol 12α-hydroxylase is an important enzyme in bile acid biosynthesis, responsible for the balance between formation of cholic acid and chenodeoxycholic acid. The enzyme has been purified to apparent homogeneity from rabbit liver (Ishida, H., Noshiro, M., Okuda, K., and Coon, M. J. (1992) J. Biol. Chem.267, 21319-21323), and we here describe the cloning and sequencing of a cDNA coding for this enzyme. After tryptic digestion of purified protein in a polyacrylamide gel, eight different peptides were isolated and sequenced. Using oligonucleotides deduced from the amino acid sequences, clones were isolated from a rabbit liver cDNA library. In addition to several overlapping clones, one full-length clone was obtained that coded for a polypeptide of 500 amino acids, corresponding to a molecular mass of 57 kDa. All of the eight peptides and the reported NH2-terminal amino acid sequence were matched against the sequence. The peptide sequence showed a 39% similarity with human prostacyclin synthase (CYP8) and 31% similarity with the rate-limiting enzyme in over-all synthesis of bile acids, the cholesterol 7α-hydroxylase (CYP7) of the rabbit. The similarity with most other sterol cytochrome P-450 hydroxylases was less. Thus, this species of cytochrome P-450 should belong to a group of its own, here denoted CYP12. Transfection of COS cells with the coding part of the cDNA resulted in a significant expression of sterol 12α-hydroxylase activity toward 7α-hydroxy-4-cholesten-3-one. Northern blotting showed that the enzyme was exclusively expressed in the liver. The major mRNA fraction in rabbit liver had a size of approximately 2.9 kilobases, and those found in rat and human liver were about 2.5 and 4.5 kilobases, respectively. Fasting of rats and mice led to a severalfold increase in both enzyme activity and mRNA levels. In contrast, starvation of rabbits had little or no stimulatory effect on enzyme activity and mRNA levels.

High dose of ascorbic acid induces cell death in mesothelioma cells

Malignant mesothelioma is an asbestos-related fatal disease with no effective cure. Recently, high dose of ascorbate in cancer treatment has been reexamined. We studied whether high dose of ascorbic acid induced cell death of four human mesothelioma cell lines. High dose of ascorbic acid induced cell death of all mesothelioma cell lines in a dose-dependent manner. We further clarified the cell killing mechanism that ascorbic acid induced reactive oxygen species and impaired mitochondrial membrane potential. In vivo experiment, intravenous administration of ascorbic acid significantly decreased the growth rate of mesothelioma tumor inoculated in mice. These data suggest that ascorbic acid may have benefits for patients with mesothelioma.

5′-,3′-Inverted Thymidine-modified Antisense Oligodeoxynucleotide Targeting Midkine ITS DESIGN AND APPLICATION FOR CANCER THERAPY

Oligodeoxynucleotides modified at both 5′- and 3′-ends with inverted thymidine (5′-,3′-inverted T) were introduced as new reagents for antisense strategies. These modifications were performed to make the oligodeoxynucleotides resistant to nucleases. The effectiveness of these oligodeoxynucleotides was evaluated in terms of inhibition of synthesis of midkine (MK), a heparin-binding growth factor, and consequent inhibition of growth of CMT-93 mouse rectal carcinoma cells. 5′-,3′-Inverted T antisense MK suppressed synthesis of MK by CMT-93 cells and their growth in culture. Furthermore, 5′-,3′-inverted T oligodeoxynucleotides exhibited less cytotoxicity and better stability than phosphorothioate oligodeoxynucleotides. When 5′-,3′-inverted T antisense MK was mixed with atelocollagen, and injected into CMT-93 tumors pregrown in nude mice, tumor growth was markedly suppressed as compared with tumors injected with sense controls. The suppressive effect of 5′-,3′-inverted T antisense MK on tumor growth was stronger than that of phosphorothioate antisense MK. These findings indicated the usefulness of inverted thymidine-modified antisense oligodeoxynucleotides as a new reagent instead of phosphorothioate-modified oligodeoxynucleotides.

Protective effect of tamoxifen on 1-methyl-4-phenylpyridine-induced hydroxyl radical generation in the rat striatum

We examined whether tamoxifen could suppress 1-methyl-4-phenylpyridine (MPP(+))-induced hydroxyl radical generation in the extracellular fluid of rat striatum, using in vivo microdialysis system. MPP(+) (5 mM) enhanced generation of hydroxyl radicals with concomitant increased efflux of dopamine. Tamoxifen (1--100 microM) dose-dependently suppressed the hydroxyl radical formation induced by MPP(+). Tamoxifen (100 microM) significantly attenuated dopamine efflux induced by MPP(+). The result in the present study is the first demonstration showing the protective effect of tamoxifen on hydroxyl radical generation induced by MPP(+) by suppressing dopamine efflux.

In utero and lactational exposure to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) affects bone tissue in rhesus monkeys.

Bone tissue is one of the target tissues for dioxins and dioxin-like compounds. Therefore, the aim of this study was to investigate effects of in utero and lactational exposure to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), on bone tissue in rhesus monkey, the most human-like experimental model available. Pregnant rhesus monkeys (Macaca mulatta; age 4-10 years) were exposed to TCDD with a total dose of 40.5-42.0 or 405-420ng/kg bodyweight by repeated subcutaneous injections starting at gestational day 20 and followed by injections every 30 days until 90 days after delivery. At a mean age of 7 years the offspring were sacrificed and the femur bone dissected. Results from peripheral Quantitative Computed Tomography (pQCT) analyses of the metaphyseal part of the femur bones in female offspring showed significant increases in trabecular bone mineral content (BMC; +84.6%, p<0.05, F-value (F)=5.9) in the low-dose treatment group compared with the controls. In the same animals, analysis of the mid-diaphyseal part revealed increases in total BMC (+21.3%, p<0.05, F=5.2) and cortical cross-sectional area (CSA; +16.4%, p<0.01, F=7.4) compared with the controls. In males, changes in biomechanical properties indicating more fragile bone were observed. Displacement at failure were significantly increased in the male low-dose group compared to the controls (+38.0%, p<0.05, F=11). The high dose of TCDD did not induce any significant changes in bone morphology. In conclusion, in utero and lactational low-dose, but not high-dose exposure to 2,3,7,8-TCDD induced disruption of bone tissue development in rhesus monkey, a result suggesting that similar effects might occur in humans also.

EGCG induces human mesothelioma cell death by inducing reactive oxygen species and autophagy.

Malignant mesothelioma is an asbestos-related fatal disease with no effective cure. We studied whether a green tea polyphenol, epigallocathechin-3-gallate (EGCG), could induce cell death in five human mesothelioma cell lines. We found that EGCG induced apoptosis in all five mesothelioma cell lines in a dose-dependent manner. We further clarified the cell killing mechanism. EGCG induced reactive oxygen species (ROS), and impaired the mitochondrial membrane potential. As treatment with ROS scavengers, catalase and tempol, significantly inhibited the EGCG-induced apoptosis, ROS is considered to be responsible for the EGCG-induced apoptosis. Further, we found that EGCG induced autophagy, and that when autophagy was suppressed by chloroquine, the EGCG-induced cell death was enhanced. Taken together, these results suggest that EGCG has a great potential for the treatment of mesothelioma by inducing apoptosis and autophagy.

A new method for the preparation of a calcium activated neutral protease highly sensitive to calcium ions

Abstract A Ca2+--activated neutral protease has been purified from chicken skeletal muscle to homogeneity by a new method which employs affinity chromatography on casein CH-Sepharose 4B. SDS polyacrylamide gel electrophoresis shows that the purified enzyme consists of a single polypeptide chain with a molecular weight of 76,000. For half-maximum activity this protease requires 50 μM Ca2+ ions and its optimum pH is 7.6. The protease is inhibited by leupeptin, antipain, E-64 and endogenous inhibitor. The purified protease is very labile upon storage; after 3 days at 4°C no detectable activity remained.

Frontal lobe dementia with abnormal cholesterol metabolism and heterozygous mutation in sterol 27-hydroxylase gene (CYP27)

Of the primary dementing disorders that cause frontotemporal dementia, the best-known is Pick disease. We report on a 44-year-old woman with progressive frontal lobe dementia and spastic paraplegia. Examination revealed increased serum levels of cholestanol with abnormal cholesterol metabolism and a heterozygous mutation of the sterol 27-hydroxylase gene (CYP27). Biochemical findings were compatible with cerebrotendinous xanthomatosis (CTX); however, the clinical manifestations were very dissimilar. To our knowledge, a symptomatic carrier of this mutation among CTX patients has not been reported. We speculate that the present patient has a previously undescribed neurodegenerative disease related to abnormal cholesterol metabolism with this heterozygous mutation.