Toru Nishinaka

Different functions between human monomeric carbonyl reductase 3 and carbonyl reductase 1

Monomeric carbonyl reductases (CBRs) are enzymes that catalyze the reduction of many endogenous and xenobiotic carbonyl compounds, including steroids and prostaglandins. There are two monomeric CBR genes in the human genome, cbr1 and cbr3, which exhibit high homology in their amino acid sequences. Human CBR1 (hCBR1) is known as prostaglandin 9-keto reductase and 15-hydroxy dehydrogenase, and regulates the metastasis of cancer cells through the regulation of prostaglandin metabolism. However, there is little information concerning the molecular and enzymatic characteristics of human CBR3 (hCBR3). The present study demonstrated the tissue and cellular localization, and catalytic activity of hCBR3. Semi-quantitative PCR revealed the ubiquitous but lower expression of hCBR3 compared with that of hCBR1. Bacterially expressed hCBR3 exhibited limited catalytic activity toward menadione, 4-benzoylpyridine, and 4-nitrobenzaldehyde. Similar results were obtained when the cell lysates of CBR-overexpressing HEK293 cells were examined. Additionally, neither the prostaglandin 9-keto reductase nor the 15-hydroxy dehydrogenase activities of hCBR3 were significant. Immunofluorescence staining revealed that ectopically expressed hCBR3 proteins were localized in the cytosol of HEK293 cells. These results suggested that hCBR3 and hCBR1 play distinct physiological roles. This study expands our understanding of the relationship between the two monomeric hCBRs and prostaglandin metabolism.

Expression of a Peroxisome Proliferator-Activated Receptor γ1 Splice Variant that Was Identified in Human Lung Cancers Suppresses Cell Death Induced by Cisplatin and Oxidative Stress

Purpose: The activation of peroxisome proliferator-activated receptor γ (PPARγ) has been implicated in the inhibition of tumor progression in lung cancers through the induction of differentiation and apoptosis. Recently, we identified a novel splice variant of human PPARγ1 (hPPARγ1) that exhibits dominant-negative activity in human tumor-derived cell lines. This study aimed to examine the expression and pathophysiologic roles of a truncated splice variant of hPPARγ1 (hPPARγ1tr) in primary human lung cancer tissues. Experimental Design: The expression and localization of hPPARγ1tr was surveyed in human primary lung cancer tissues using immunohistochemistry and Western blot analysis. Using transfectants stably expressing wild-type hPPARγ1 (hPPARγ1wt) and hPPARγ1tr, we also analyzed the pathophysiologic roles of hPPARγ1tr. Results: We showed that PPARγ is expressed predominantly in the nucleus of nontumorous tissues, whereas it is present in both the nucleus and the cytoplasm of tumorous tissues in squamous cell carcinoma (SCC) of the lung. Western blot analysis confirmed the presence of PPARγ1tr in primary lung SCC tissue but not in nontumorous tissue. Expression of PPARγ1tr in Chinese hamster ovary cells attenuated their susceptibility to cell death induced by oxidative stress or cisplatin, whereas their susceptibility was completely recovered by down-regulation of PPARγ1tr with small interfering RNA. Conclusions: hPPARγ1tr is expressed strongly in tumorous tissues of primary human lung SCC and its overexpression renders transfected cells more resistant to chemotherapeutic drug- and chemical-induced cell death. These data suggest that the decreased drug sensitivity of PPARγ1tr-expressing cells may be associated with increased tumor aggressiveness and poor clinical prognosis of patients.

Regulation of human carbonyl reductase 1 (CBR1, SDR21C1) gene by transcription factor Nrf2

Monomeric carbonyl reductase 1 (CBR1, SDR21C1) is a member of the short-chain dehydrogenase/reductase superfamily and is involved in the metabolism of anthracycline anti-cancer drugs, prostaglandins, and isatin, which is an endogenous inhibitor of monoamine oxidases. Additionally, cancer progression may be partly regulated by CBR1. In the present study, we screened more than 10 drugs for the induction of the human CBR1 gene to investigate its regulation. Of the drugs, butylated hydroxyanisole (BHA) was found to be an inducer. BHA induced the mRNA and protein expression of CBR1 in hepatoma HepG2 cells. In a luciferase reporter gene assay, the promoter region between -2062 bp and the transcription start site of CBR1 was also activated by BHA. The transcription factor Nrf2 is known to be activated by BHA. There are 2 anti-oxidant responsive elements (ARE) that are bound by Nrf2 in this region. Mutation analyses revealed that one of the AREs participates in the gene regulation of CBR1 by Nrf2. Electrophoretic mobility shift assay revealed that Nrf2 binds the site. Moreover, to determine whether the functional ARE of CBR1 is conserved with the promoter region of homologues in other species, the nucleotide sequences of the functional AREs of the Chcr1 and Chcr2 genes, which are the Chinese hamster homologues of CBR1, were determined. The region has 2 AREs, and these genes were also induced by the forced expression of Nrf2 (cotransfection of pNrf2) in the luciferase reporter gene assay. In conclusion, Nrf2 is a novel transcriptional regulator of CBR1 genes in humans and the Chinese hamster. Because the regulation of CBR1 appears to be important for diseases, the induction of CBR1 by Nrf2 may be a therapeutic target.

Structure-activity relationship of flavonoids as potent inhibitors of carbonyl reductase 1 (CBR1).

Human carbonyl reductase 1 (CBR1), a member of the short-chain dehydrogenase/reductase superfamily, reduces a variety of carbonyl compounds including therapeutic drugs. CBR1 is involved in the reduction of the anthracycline anticancer drugs to their less anticancer C-13 hydroxy metabolites, which are cardiotoxic. CBR1 inhibitors are thought to be promising agents for adjuvant therapy with twofold beneficial effect in prolonging the anticancer efficacy of the anthracyclines while decreasing cardiotoxicity, a side effect of the drugs. In this study, we evaluated 27 flavonoids for their inhibitory activities of CBR1 in order to explore the structure-activity relationship (SAR). Among them, luteolin (2-(3,4-dihydroxyphenyl)-5,7-dihydroxy-4H-1-benzopyran-4-one) showed the most potent inhibition (IC5095nM), which is also more potent compared to all known classes of CBR1 inhibitors. The inhibition of luteolin was noncompetitive with respect to the substrate in the NADPH-dependent reduction direction, but CBR1 exhibited moderate NADP(+)-dependent dehydrogenase activity for some alicyclic alcohols, in which the luteolin inhibition was competitive with respect to the alcohol substrate (Ki59nM). The SAR of the flavonoids indicated that the 7-hydroxy group of luteolin was responsible for the potent inhibition of CBR1. The molecular docking of luteolin in CBR1-NADPH complex showed that theflavonoid binds to the substrate-binding cleft, in which its 7-hydroxy group formed a H-bond with main-chain oxygen of Met234, in addition to H-bond interactions (of its 5-hydroxy and 4-carbonyl groups with catalytically important residues Tyr193 and/or Ser139) and a π-stacking interaction (between its phenyl ring and Trp229).

Relationship between aging and dosage of warfarin : The current status of warfarin anticoagulant therapy for Japanese outpatients in a department of cardiovascular medicine

BACKGROUND Oral anticoagulant therapy with warfarin is essential for the optimal prophylaxis of thrombosis and embolism in many vascular disorders whose prevalence is high among elderly patients. Elderly Caucasians show increased sensitivity to oral anticoagulants. Although the Japanese are more sensitive to warfarin therapy than Caucasians, the relationship between age and warfarin dosage in the Japanese has not been investigated in detail. Here, we investigated this relationship in Japanese patients. METHODS The subjects comprised 102 outpatients (28-87 years) who showed a stable anticoagulant effect (prothrombin time expressed in terms of the international normalized ratio [PT-INR] between 1.6 and 2.6) following long-term warfarin therapy at the Department of Cardiovascular Medicine, National Hospital Organization Hakodate Hospital. The relationship between age, PT-INR, and daily warfarin dose was retrospectively investigated. RESULTS The mean values of the characteristics of all the patients were as follows: age, 70.7 (+/-10.5) years; daily dose of warfarin, 2.68 (+/-0.95)mg; PT-INR, 1.99 (+/-0.24). The PT-INR was not correlated with increasing age. The daily dosage of warfarin and the dosage adjusted according to the PT-INR (dose/PT-INR), wherein the lower values indicate a higher sensitivity to warfarin therapy, were significantly inversely correlated with age. In the high PT-INR (PT-INR>2.1) group, no significant difference was observed between the older and younger patients with regard to any characteristics. Conversely, in the low PT-INR (1.6 ≤ PT-INR ≤ 2.1) group, the dose/PT-INR was significantly reduced in the older patients (≥ 65 years); however, no significant difference was observed in the PT-INR value. CONCLUSIONS Both age and the controlled PT-INR value are factors responsible for the increasing sensitivity to warfarin in Japanese patients under optimal anticoagulant therapy. Since elderly Japanese patients with low PT-INR values are especially sensitive to warfarin, greater caution should be exercised while determining the dosage schedule in such patients.

Regulation of transforming growth factor β1-dependent aldose reductase expression by the Nrf2 signal pathway in human mesangial cells.

Aldose reductase (AR) is a key enzyme in the alternative glucose metabolism pathway, the polyol pathway. To date, AR is known to be involved in several secondary complications of diabetes and various kidney diseases. The goal of this study was to elucidate how the Nrf2-anti-oxidant response element (ARE) signal pathway plays a role in TGFβ1s regulation of AR expression in human renal mesangial cells (HRMCs). As an in vitro model system, HRMCs were used to investigate AR mRNA by qPCR, protein by Western blot and enzymatic activity by spectrophotometric assay. The ability of TGFβ1 to induce reactive oxygen species (ROS) in cells was measured by electron-spin resonance (ESR) trapping method. Reporter assays were used to test the activity of the AR promoter region, and ChIP was employed to test the direct binding of Nrf2 with the endogenous AR promoter. Treatment of HRMCs with TGFβ1 up-regulated the expression of AR mRNA, protein, and activity level. Additionally, TGFβ1 rapidly increased cellular ROS levels, which in turn activated the Nrf2-ARE pathway. Either inhibition of ROS production or knockdown of Nrf2 in HRMCs decreased the TGFβ1-induction of AR expression. Nrf2 regulated AR luciferase activity specifically via two AREs within the AR promoter, and bound directly to the endogenous AR promoter. Furthermore, the TGFβ1-mediated expression of AR required Nrf2 and was significantly abrogated in Nrf2-/- cells. These data show the regulation of AR by TGFβ1 is induced by TGFβ1 stimulation of ROS, which activates the Nrf2-ARE pathway allowing Nrf2 to directly increase AR expression in HRMCs.

Up-Regulation of Carbonyl Reductase 1 Renders Development of Doxorubicin Resistance in Human Gastrointestinal Cancers

Doxorubicin (DOX) is widely used for the treatment of a wide range of cancers such as breast and lung cancers, and malignant lymphomas, but is generally less efficacious in gastrointestinal cancers. The most accepted explanation for the DOX refractoriness is its resistance development. Here, we established DOX-resistant phenotypes of human gastric MKN45 and colon LoVo cells by continuous exposure to incremental concentrations of the drug. While the parental MKN45 and LoVo cells expressed carbonyl reductase 1 (CBR1) highly and moderately, respectively, the gain of DOX resistance further elevated the CBR1 expression. Additionally, the DOX-elicited cytotoxicity was lowered by overexpression of CBR1 and inversely strengthened by knockdown of the enzyme using small interfering RNA or pretreating with the specific inhibitor quercetin, which also reduced the DOX refractoriness of the two resistant cells. These suggest that CBR1 is a key enzyme responsible for the DOX resistance of gastrointestinal cancer cells and that its inhibitor is useful in the adjuvant therapy. Although CBR1 is known to metabolize DOX to a less toxic anticancer metabolite doxorubicinol, its overexpression in the parental cells hardly show significant reductase activity toward low concentration of DOX. In contrast, the overexpression of CBR1 increased the reductase activity toward an oxidative stress-derived cytotoxic aldehyde 4-oxo-2-nonenal. The sensitivity of the DOX-resistant cells to 4-oxo-2-nonenal was lower than that of the parental cells, and the resistance-elicited hyposensitivity was almost completely ameliorated by addition of the CBR1 inhibitor. Thus, CBR1 may promote development of DOX resistance through detoxification of cytotoxic aldehydes, rather than the drugs metabolism.

Importance of the substrate-binding loop region of human monomeric carbonyl reductases in catalysis and coenzyme binding.

AIMS Monomeric carbonyl reductase 1 (CBR1) and 3 (CBR3) are members of the short-chain dehydrogenase/reductase superfamily, and metabolize endogenous and xenobiotic compounds using NADPH as a coenzyme. CBR3 exhibits a higher K(m) value toward NADPH and more limited carbonyl reductase activities than CBR1, although they are highly homologous to each other in amino acid sequence levels. In the present study, we investigated the origin of the different properties of the enzymes by analyses using several chimeric enzymes. MAIN METHODS Harr-plot analysis of the amino acid sequences was conducted and as a result, two low-identity regions between human CBR1 and CBR3 were found: these were designated as the N-terminal low-identity region (LirN) and the C-terminal low-identity region (LirC; the substrate-binding region). We genetically constructed chimeric enzymes while focusing on these regions. KEY FINDINGS Chimeric CBR1 possessing LirN of CBR3 (CBR1LirN3) exhibited CBR1-like activities but a low coenzyme affinity probably due to a structural alteration in a micro domain, whereas chimeric CBR1 including LirC of CBR3 (CBR1LirC3) was enzymatically similar to CBR3. Furthermore, CBR3LirC1 was similar to CBR1 in both enzymatic activities and coenzyme binding. SIGNIFICANCE These results suggested that LirC, i.e., the substrate-binding loop region, is the origin of the difference between human CBR1 and CBR3 in both catalytic and coenzyme-binding properties.

Transcription factor AP-1 regulates TGF-β1-induced expression of aldose reductase in cultured human mesangial cells

Aim:  The previous studies demonstrated that transforming growth factor‐β1 (TGF‐β1) could upregulate the expression of aldose reductase (AR). The aim of this study is to clarify (investigate) the mechanism of TGF‐β1‐induced AR expression.

Down-regulation of aldo–keto reductase AKR1B10 gene expression by a phorbol ester via the ERK/c-Jun signaling pathway

AKR1B10 is a human member of the aldo-keto reductase (AKR) superfamily, and is considered to be a tumor biomarker because its expression is known to be significantly induced in the cells of various cancers such as lung non-small-cell carcinoma and hepatocellular carcinoma. However, the mechanisms underlying the regulation of its gene remain unclear. In the present study, we demonstrated that the phorbol ester, 12-O-tetradecanoyl phorbol 13-acetate (TPA), down-regulated the expression of the AKR1B10 gene in the human lung cancer cell line, A549. The treatment of A549 cells with TPA for 24h significantly reduced the mRNA levels, protein levels, and promoter activity of AKR1B10 as well as the growth of A549 cells. TPA induced the phosphorylation of the MAP kinase, ERK, and U0126, an inhibitor of the MAP kinase kinase, MEK1, blocked the down-regulation of AKR1B10 by TPA, indicating that the MAP kinase ERK plays a role in regulating the expression of AKR1B10. TPA also induced c-jun gene expression in an ERK-dependent manner. The co-introduction of the c-Jun protein resulted in a decrease in the mRNA levels and promoter activity of AKR1B10 as well as A549 cell proliferation. These results suggested that the ERK/c-Jun signaling pathway may play an important role in the TPA-triggered down-regulation of AKR1B10 gene expression.