Carbonyl Reductase 1 Research Articles

Inhibitory effect of carbonyl reductase 1 on ovarian cancer growth via tumor necrosis factor receptor signaling.

We investigated the mechanisms of the inhibitory effect of carbonyl reductase 1 (CR1) on ovarian cancer growth mediated by the activation of the tumor necrotic factor receptor (TNFR) pathway. OVCAR-3 and TOV21G cells overexpressing CR1 were constructed by transfecting them with CR1 cDNA by lipofection. CR1-overexpressing and control OVCAR-3 and TOV21G cells were injected subcutaneously into nude mice and the tumor growth was compared between the two groups for 3-4 weeks. The expression of TNFR1 and TNFR2 in tumors was examined immunohistochemically at the end of the experiment. Expression levels of caspase-8 and -3 activated by TNFR1, c-Jun activated by TNFR2, and NF-κB activated by both TNFR1 and TNFR2 were determined using immunohistochemistry and western blot analysis. Tumor growth was significantly suppressed in mice injected with CR1-overexpressing cells. Tumor volume in the CR1 induction group decreased temporarily until 2 weeks. Tumor cell membranes in both CR1 induction and control groups were positive for TNFR1 expression; however, total protein levels did not differ between the two groups. TNFR-2 expression was comparatively weak in both groups. The expression of NF-κB and c-Jun was weaker in the CR1 induction group than in control. In contrast, caspase-8 and -3 expression was higher in the CR1 induction group. Furthermore, the number of apoptotic cells was significantly greater in tumors that appeared after injections of both types of CR1-overexpressing cells than in those of control cancer cells. These results suggest that CR1 induces apoptosis by activating the caspase pathway via binding to TNFR1.

Bioactivation of loxoprofen to a pharmacologically active metabolite and its disposition kinetics in human skin.

Loxoprofen (LX) is a prodrug-type non-steroidal anti-inflammatory drug which is used not only as an oral drug but also as a transdermal formulation. As a pharmacologically active metabolite, the trans-alcohol form of LX (trans-OH form) is generated after oral administration to humans. The objectives of this study were to evaluate the generation of the trans-OH form in human in vitro skin and to identify the predominant enzyme for its generation. In the permeation and metabolism study using human in vitro skin, both the permeation of LX and the formation of the trans-OH form increased in a time- and dose-dependent manner after the application of LX gel to the skin. In addition, the characteristics of permeation and metabolism of both LX and the trans-OH form were examined by a mathematical pharmacokinetic model. The Km value was calculated to be 10.3 mm in the human in vitro skin. The predominant enzyme which generates the trans-OH form in human whole skin was identified to be carbonyl reductase 1 (CBR1) by immunodepletion using the anti-human CBR1 antibody. The results of the enzyme kinetic study using the recombinant human CBR1 protein demonstrated that the Km and Vmax values were 7.30 mm and 402 nmol/min/mg protein, respectively. In addition, it was found that no unknown metabolites were generated in the human in vitro skin. This is the first report in which LX is bioactivated to the trans-OH form in human skin by CBR1. Copyright © 2015 John Wiley & Sons, Ltd.

Redox proteomics identification of specifically carbonylated proteins in the hippocampi of triple transgenic Alzheimer's disease mice at its earliest pathological stage

Alzheimer's disease (AD) is the most common cause of dementia in the elderly population. Attempts to develop therapies for the treatment of the late stage AD have been unsuccessful. Increasing evidences indicate that oxidative stress is an early event of neurodegeneration, however the pathogenic mechanism of AD remains unclarified. In the present study, slot-blot analysis was used to determine the levels of protein carbonyls in the hippocampi of 3-month-old triple transgenic AD mice (3×Tg-AD). The increased levels of protein carbonyls were observed in the hippocampi of 3×Tg-AD mice as compared to the non-transgenic controls (non-Tg). Using a redox-proteomic approach, twelve proteins were found to be significantly altered in the levels of protein carbonyls in the hippocampus. These proteins are crucial in energy metabolism, protein folding, cell structure, signal transduction and excitotoxicity. Immunoprecipitation and Western blot were used to validate two proteins identified by the redox proteomics. In addition, increased expression level of carbonyl reductase 1 (CBR1) was observed in the hippocampi of 3×Tg-AD mice. These results demonstrate that significant protein carbonylation occurs early in the 3-month-old 3×Tg-AD mice, which support the viewpoint that oxidative stress is an early event in AD progression. Biological significanceIn this study, we have observed increased levels of protein carbonyls in the hippocampi of 3×Tg-AD mice before the appearance of Aβ plaques and neurofibrillary tangles (NFTs). By redox proteomics, twelve specifically carbonylated proteins were identified. Among them, alpha-enolase (ENO1) and glutamine synthetase (GS) were identified as the common targets of oxidation in the brains of 3×Tg-AD mice, mild cognitive impairment (MCI) sufferers and AD patients. For the first time, the oxidation of t-complex protein 1 subunit epsilon (CCT5) and protein disulfide-isomerase A3 (PDIA3) were reported to be associated with AD. These results indicated that the combination of monoclonal anti-DNP antibody with digital imaging system could enhance the specificity and accuracy of redox proteomics analysis. Those data support the viewpoint that oxidative stress occurs at the early pathological stage of AD. In addition, this paper provides new information for understanding the pathological process of AD and for developing more appropriate therapies to intervene AD progression.

Involvement of microsomal NADPH-cytochrome P450 reductase in metabolic reduction of drug ketones.

Recently, it was found that the carbonyl group of 1-[3-(4-phenoxyphenoxy)-2-oxopropyl]indole-5-carboxylic acid (5), an inhibitor of the pro-inflammatory enzyme cytosolic phospholipase A2 α, is easily reduced by rat liver S9 fractions in vitro. Determination of the inhibitory potency of certain putative inhibitors of carbonyl reducing enzymes on the transformation of the ketone derivative 5 to its alcohol 6 by recombinant microsomal NADPH-cytochrome P450 reductase and by recombinant cytosolic carbonyl reductase-1 now reveals that these compounds show a lack of specificity for these two enzymes in part. Thus, an assignment of the roles of different carbonyl reductases in metabolic keto reduction by the use of inhibitors is problematic. In addition, the ability of NADPH-cytochrome P450 reductase and carbonyl reductase-1 to reduce the ketone groups of the drugs haloperidol and daunorubicin was examined. Under the conditions applied, a pronounced reductive metabolism was only observed for daunorubicin in the presence of microsomal NADPH-cytochrome P450 reductase. Similarly, in rat liver S9 fractions a marked reduction of daunorubicin was seen, while haloperidol was only slightly metabolized to its alcohol. After separation of the S9 homogenate into a microsomal and a cytosolic fraction, it became evident that the ketone groups of daunorubicin, haloperidol and compound 5 were mainly reduced by cytosolic enzymes. However, since microsomes also catalysed these carbonyl reductions to some extent, it can be concluded that microsomal NADPH-cytochrome P450 reductase can contribute to metabolic keto reductions in xenobiotics. Copyright © 2015 John Wiley & Sons, Ltd.

Tat-CBR1 inhibits inflammatory responses through the suppressions of NF-κB and MAPK activation in macrophages and TPA-induced ear edema in mice

Human carbonyl reductase 1 (CBR1) plays a crucial role in cell survival and protects against oxidative stress response. However, its anti-inflammatory effects are not yet clearly understood. In this study, we examined whether CBR1 protects against inflammatory responses in macrophages and mice using a Tat-CBR1 protein which is able to penetrate into cells. The results revealed that purified Tat-CBR1 protein efficiently transduced into Raw 264.7 cells and inhibited lipopolysaccharide (LPS)-induced cyclooxygenase-2 (COX-2), nitric oxide (NO) and prostaglandin E2 (PGE2) expression levels. In addition, Tat-CBR1 protein leads to decreased pro-inflammatory cytokine expression through suppression of nuclear transcription factor-kappaB (NF-κB) and mitogen activated protein kinase (MAPK) activation. Furthermore, Tat-CBR1 protein inhibited inflammatory responses in 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced skin inflammation when applied topically. These findings indicate that Tat-CBR1 protein has anti-inflammatory properties in vitro and in vivo through inhibition of NF-κB and MAPK activation, suggesting that Tat-CBR1 protein may have potential as a therapeutic agent against inflammatory diseases.

Protective effect of 23-hydroxybetulinic acid on doxorubicin-induced cardiotoxicity: a correlation with the inhibition of carbonyl reductase-mediated metabolism.

The clinical use of doxorubicin, an effective anticancer drug, is severely hampered by its cardiotoxicity. 23-Hydroxybetulinic acid (23-HBA), isolated from Pulsatilla chinensis, enhances the anticancer effect of doxorubicin while simultaneously reducing its cardiac toxicity, but does not affect the concentration of doxorubicin in the plasma and heart. As the metabolite doxorubicinol is more potent than doxorubicin at inducing cardiac toxicity, in the present study we aimed to clarify the role of doxorubicinol in the protective effect of 23-HBA. Doxorubicin was administered to mice for two weeks in the presence or absence of 23-HBA. The heart pathology, function, myocardial enzymes and accumulation of doxorubicin and doxorubicinol were then analysed. A cellular pharmacokinetic study of doxorubicin and doxorubicinol, carbonyl reductase 1 (CBR1) interference and molecular docking was performed in vitro. 23-HBA alleviated the doxorubicin-induced cardiotoxicity in mice, and this was accompanied by inhibition of the metabolism of doxorubicin and reduced accumulation of doxorubicinol selectively in hearts. In H9c2 cells, the protective effect of 23-HBA was shown to be closely associated with a decreased rate and extent of accumulation of doxorubicinol in mitochondria and nuclei. siRNA and docking analysis demonstrated that CBR1 has a crucial role in doxorubicin-mediated cardiotoxicity and 23-HBA inhibits this metabolic pathway. Inhibition of CBR-mediated doxorubicin metabolism might be one of the protective mechanisms of 23-HBA against doxorubicin-induced cardiotoxicity. The present study provides a new research strategy guided by pharmacokinetic theory to elucidate the mechanism of drugs with unknown targets.

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 drug's metabolism.

Synthesis of 8-hydroxy-2-iminochromene derivatives as selective and potent inhibitors of human carbonyl reductase 1

Human carbonyl reductase 1 (CBR1), a member of the short-chain dehydrogenase/reductase superfamily, reduces anthracycline anticancer drugs to their less potent anticancer C-13 hydroxy metabolites, which are linked with pathogenesis of cardiotoxicity, a side effect of the drugs. CBR1 inhibitors are thought to be promising agents for adjuvant therapy with a twofold beneficial effect in prolonging the anticancer efficacy of the anthracyclines while decreasing cardiotoxicity. In order to search for new potential inhibitors of CBR1, we synthesized a series of des-methoxyphenyl derivatives of (Z)-2-(4-methoxyphenylimino)-7-hydroxy-N-(pyridin-2-yl)-2H-chromene-3-carboxamide (1) that was developed previously as a potent inhibitor of aldo-keto reductase (AKR) 1B10 and AKR1B1. Among the newly synthesized inhibitors, 8-hydroxy-2-imino-2H-chromene-3-carboxylic acid (2-chlorophenyl)amide (13h) was the most potent competitive inhibitor of CBR1, showing a Ki value of 15 nM. 13h also showed high selectivity to CBR1 over its isozyme CBR3 and other enzymes with CBR activity (AKR1B1, AKR1B10, AKR1C1, AKR1C2, AKR1C4, DXCR and DHRS4). Furthermore, 13h inhibited the cellular metabolism by CBR1 at its concentration of 4 μM. The structure-activity relationship of the derivatives, site-directed mutagenesis of putative binding residues (Met141 and Trp229) and molecular docking of 13h in CBR1 revealed that the interactions of 13h with the substrate-binding residues (Ser139, Met141, Tyr193 and Trp229) are important for the tight binding.

Decreased expression of carbonyl reductase 1 promotes ovarian cancer growth and proliferation.

Carbonyl reductase 1 (CBR1) expression level is related to tumor progression. Decreased CBR1 expression is associated with poor prognosis in ovarian cancer. We investigated the relationship between CBR1 expression level and malignant potential of ovarian cancer. OVCAR‑3 cells overexpressing CBR1 or knocked down for CBR1 were obtained by transfecting CBR1 plasmid DNA (pDNA) or small interfering RNA (siRNA) by electroporation. In vitro cell proliferation and invasion were compared between the two cell types. Subcutaneous CBR1‑overexpressed OVCAR‑3 cells (n=10) and wild‑type ones (n=5) were injected into nude mice. The CBR1 siRNA was then injected twice a week into five of the 10 CBR1‑overexpressed OVCAR‑3 tumors. Tumor growth and metastatic behavior were observed 3 weeks after cell transplantation. Cell proliferation significantly decreased in CBR1‑overexpressed cells as compared to the control, whereas cell proliferation and invasion significantly increased in CBR1‑suppressed cells as compared to the control. The size of the CBR1 siRNA‑injected tumors (n=5) increased significantly as compared to the other two groups (n=5 for each group). The number of metastatic foci in the lungs was significantly higher in mice injected with CBR1 siRNA (7.0±2.0) compared to CBR1‑overexpressed and wild‑type tumors (0 and 2.0±2.0, respectively). Western blot analysis showed that, while vascular endothelial growth factor (VEGF)‑C expression was stable in the CBR1‑siRNA‑injected tumors, E‑cadherin expression was decreased, whereas matrix metalloproteinase (MMP)‑9 was increased in CBR1‑siRNA‑injected tumors compared to the other two groups. These results showed that CBR1 decreases promoted tumor proliferation and growth as well as invasion and metastasis, suggesting that CBR1 has potential to become a new candidate for molecular targeting therapy.

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).

Metabolism of doxorubicin to the cardiotoxic metabolite doxorubicinol is increased in a mouse model of chronic glutathione deficiency: A potential role for carbonyl reductase 3

Doxorubicin is highly effective at inducing DNA double-strand breaks in rapidly dividing cells, which has led to it being a widely used cancer chemotherapeutic. However, clinical administration of doxorubicin is limited by off-target cardiotoxicity, which is thought to be mediated by doxorubicinol, the primary alcohol metabolite of doxorubicin. Carbonyl reductase 1 (CBR1), a well-characterized monomeric enzyme present at high basal levels in the liver, is known to exhibit activity toward doxorubicin. Little is known about a closely related enzyme, carbonyl reductase 3 (CBR3), which is present in the liver at low basal levels but is highly inducible by the transcription factor Nrf2. Genetic polymorphisms in CBR3, but not CBR1, are associated with differential cardiac outcomes in doxorubicin treated pediatric patients. Cbr3 mRNA and CBR3 protein are highly expressed in the livers of Gclm−/− mice (a mouse model of glutathione deficiency) relative to wild type mice. In the present study, we first investigated the ability of CBR3 to metabolize doxorubicin. Incubations of doxorubicin and purified recombinant murine CBR3 (mCBR3) were analyzed for doxorubicinol formation using HPLC, revealing for the first time that doxorubicin is a substrate of mCBR3. Moreover, hepatocytes from Gclm−/− mice produced more doxorubicinol than Gclm+/+ hepatocytes. In addition, differentiated rat myoblasts (C2C12 cells) co-cultured with primary Gclm−/− murine hepatocytes were more sensitive to doxorubicin-induced cytostasis/cytotoxicity than incubations with Gclm+/+ hepatocytes. Our results indicate a potentially important role for CBR3 in doxorubicin-induced cardiotoxicity. Because there is likely to be variability in hepatic CBR3 activity in humans (due to either genetic or epigenetic influences on its expression), these data also suggest that inhibition of CBR3 may provide protection from doxorubicinol cardiotoxicity.

Effect of standardized cranberry extract on the activity and expression of selected biotransformation enzymes in rat liver and intestine.

The use of dietary supplements containing cranberry extract is a common way to prevent urinary tract infections. As consumption of these supplements containing a mixture of concentrated anthocyanins and proanthocyanidins has increased, interest in their possible interactions with drug-metabolizing enzymes has grown. In this in vivo study, rats were treated with a standardized cranberry extract (CystiCran®) obtained from Vaccinium macrocarpon in two dosage schemes (14 days, 0.5 mg of proanthocyanidins/kg/day; 1 day, 1.5 mg of proanthocyanidins/kg/day). The aim of this study was to evaluate the effect of anthocyanins and proanthocyanidins contained in this extract on the activity and expression of intestinal and hepatic biotransformation enzymes: cytochrome P450 (CYP1A1, CYP1A2, CYP2B and CYP3A), carbonyl reductase 1 (CBR1), glutathione-S-transferase (GST) and UDP-glucuronosyl transferase (UGT). Administration of cranberry extract led to moderate increases in the activities of hepatic CYP3A (by 34%), CYP1A1 (by 38%), UGT (by 40%), CBR1 (by 17%) and GST (by 13%), while activities of these enzymes in the small intestine were unchanged. No changes in the relative amounts of these proteins were found. Taken together, the interactions of cranberry extract with simultaneously administered drugs seem not to be serious.

Prostaglandin pathway gene expression in human placenta, amnion and choriodecidua is differentially affected by preterm and term labour and by uterine inflammation

BackgroundElucidation of the biochemical pathways involved in activation of preterm and term human labour would facilitate the development of effective management and inform judgements regarding the necessity for preterm tocolysis and post-term induction. Prostaglandins act at all stages of human reproduction, and are potentially activators of labour.MethodsExpression of 15 genes involved in prostaglandin synthesis, transport and degradation was measured by qPCR using tissue samples from human placenta, amnion and choriodecidua at preterm and full-term vaginal and caesarean delivery. Cellular localisation of eight prostaglandin pathway proteins was determined by immunohistochemistry.ResultsExpression of prostaglandin pathway genes was differentially affected by factors including gestational age at delivery, and the incidence and duration of labour. Chorioamnionitis/deciduitis was associated with upregulation of PTGS2 (prostaglandin-endoperoxide synthase 2 (prostaglandin G/H synthase and cyclooxygenase)), along with the inflammatory genes IL8 (interleukin 8), S100A8 (S100 calcium binding protein A8) and TLR2 (toll-like receptor 2), in amnion and choriodecidua, and with downregulation of CBR1 (carbonyl reductase 1) and HPGD (hydroxyprostaglandin dehydrogenase 15-(NAD)) in choriodecidua. Protein localisation differed greatly between the various maternal and fetal cell types.ConclusionsPreterm and term labour are associated with distinct prostaglandin pathway expression profiles; inflammation provokes specific changes, unrelated to the presence of labour; spontaneous and induced term labour are indistinguishable.

Auto-amplification system for prostaglandin F2α in bovine corpus luteum.

The bovine corpus luteum (CL) is hypothesized to utilize a local auto-amplification system for prostaglandin (PG) F2α production. The objective of the present study was to determine if such a PGF2α auto-amplification system exists in the bovine CL, and if so, which factors regulate it. PGF2α significantly stimulated intra-luteal PGF2α production in all luteal phases, but did not affect PGE2 production. The stimulatory effect of exogenous PGF2α on CL PGF2α production was lower at the early luteal phase. Indomethacin, an inhibitor of prostaglandin-endoperoxide synthase (PTGS), significantly suppressed the PGF2α-stimulated PGF2α production by luteal tissue, indicating that the PGF2α in the medium was of luteal origin. Consistent with these secreted-PGF2α profiles, PGF2α receptor (PTGFR) protein expression was higher during the mid and late luteal phases than at early and developing luteal phases. Treatment of cultured bovine luteal cells obtained from the mid-luteal phase with PGF2α (1 µM) significantly increased the expressions of PTGS2, PGF synthase (PGFS), and carbonyl reductase1 (CBR1) at 24 hr post-treatment. Together, these results suggest the presence of a local auto-amplification system for PGF2α mediated by PTGS2, PGFS, and CBR1 in the bovine CL, which may play an important role in luteolysis.

Induction of PGF2α Synthesis by Cortisol Through GR Dependent Induction of CBR1 in Human Amnion Fibroblasts

Abundant evidence indicates a pivotal role of prostaglandin F2α (PGF2α) in human parturition. Both the fetal and maternal sides of the fetal membranes synthesize PGF2α. In addition to the synthesis of PGF2α from PGH2 by PGF synthase (PGFS), PGF2α can also be converted from PGE2 by carbonyl reductase 1 (CBR1). Here, we showed that there was concurrent increased production of cortisol and PGF2α in association with the elevation of CBR1 in human amnion obtained at term with labor versus term without labor. In cultured primary human amnion fibroblasts, cortisol (0.01-1μM) increased PGF2α production in a concentration-dependent manner, in parallel with elevation of CBR1 levels. Either siRNA-mediated knockdown of glucocorticoid receptor (GR) expression or GR antagonist RU486 attenuated the induction of CBR1 by cortisol. Chromatin immunoprecipitation (ChIP) showed an increased enrichment of both GR and RNA polymerase II to CBR1 promoter. Knockdown of CBR1 expression with siRNA or inhibition of CBR1 activity with rutin decreased both basal and cortisol-stimulated PGF2α production in human amnion fibroblasts. In conclusion, CBR1 may play a critical role in PGF2α synthesis in human amnion fibroblasts, and cortisol promotes the conversion of PGE2 into PGF2α via GR-mediated induction of CBR1 in human amnion fibroblasts. This stimulatory effect of cortisol on CBR1 expression may partly explain the concurrent increases of cortisol and PGF2α in human amnion tissue with labor, and these findings may account for the increased production of PGF2α in the fetal membranes prior to the onset of labor.

Neuroprotective effects of PEP-1-carbonyl reductase 1 against oxidative-stress-induced ischemic neuronal cell damage

Human carbonyl reductase 1 (CBR1) is a member of the NADPH-dependent short-chain dehydrogenase/reductase superfamily that is known to play an important role in neuronal cell survival via its antioxidant function. Oxidative stress is one of the major causes of degenerative disorders including ischemia. However, the role CBR1 plays with regard to ischemic injury is as yet poorly understood. Protein transduction domains such as PEP-1 are well known and now commonly used to deliver therapeutic proteins into cells. In this study, we prepared PEP-1–CBR1 protein and examined whether it protects against oxidative-stress-induced neuronal cell damage. PEP-1–CBR1 protein was efficiently transduced into hippocampal neuronal HT-22 cells and protected against hydrogen peroxide (H2O2)-induced neuronal cell death. Transduced PEP-1–CBR1 protein drastically inhibited H2O2-induced reactive oxygen species production, the oxidation of intracellular macromolecules, and the activation of mitogen-activated protein kinases, as well as cellular apoptosis. Furthermore, we demonstrated that transduced PEP-1–CBR1 protein markedly protected against neuronal cell death in the CA1 region of the hippocampus resulting from ischemic injury in an animal model. In addition, PEP-1–CBR1 protein drastically reduced activation of glial cells and lipid peroxidation in an animal model. These results indicate that PEP-1–CBR1 protein significantly protects against oxidative-stress-induced neuronal cell death in vitro and in vivo. Therefore, we suggest that PEP-1–CBR1 protein may be a therapeutic agent for the treatment of ischemic injuries as well as oxidative-stress-induced cell damage and death.

Resveratrol reduces the hypoxia-induced resistance to doxorubicin in breast cancer cells.

Resveratrol (3,4',5-trihydroxy-trans-stilbene) is known to enhance the cytotoxicity of the anticancer drug doxorubicin. On the other hand, breast cancer MCF-7 cells acquire resistance to doxorubicin under hypoxic conditions. In this study, we investigated the effect of resveratrol on hypoxia-induced resistance to doxorubicin in MCF-7 cells. Resveratrol and its derivative 3,5-dihydroxy-4'-methoxy-trans-stilbene, but not 3,5-dimethoxy-4'-hydroxy-trans-stilbene, cancelled hypoxia-induced resistance to doxorubicin at a concentration of 10 μM. Carbonyl reductase 1 (CBR1) catalyzes the conversion of doxorubicin to its metabolite doxorubicinol, which is much less effective than doxorubicin. Hypoxia increased the expression of CBR1 at both mRNA and protein levels, and knockdown of CBR1 inhibited hypoxia-induced resistance to doxorubicin in MCF-7 cells. Knockdown of hypoxia-inducible factor (HIF)-1α repressed the hypoxia-induced expression of CBR1. Resveratrol repressed the expression of HIF-1α protein, but not HIF-1α mRNA, and decreased hypoxia-activated HIF-1 activity. Resveratrol repressed the hypoxia-induced expression of CBR1 at both mRNA and protein levels. Likewise, 3,5-dihydroxy-4'-methoxy-trans-stilbene decreased the hypoxia-induced expression of CBR1 protein, but not 3,5-dimethoxy-4'-hydroxy-trans-stilbene. Furthermore, resveratrol decreased the expression of HIF-1α protein even in the presence of the proteasome inhibitor MG132 in hypoxia. Theses results indicate that in MCF-7 cells, HIF-1α-increased CBR1 expression plays an important role in hypoxia-induced resistance to doxorubicin and that resveratrol and 3,5-dihydroxy-4'-methoxy-trans-stilbene decrease CBR1 expression by decreasing HIF-1α protein expression, perhaps through a proteasome-independent pathway, and consequently repress hypoxia-induced resistance to doxorubicin.

Carbonyl reductase 1 is an essential regulator of skeletal muscle differentiation and regeneration

It is well established that reactive oxygen species (ROS) are essential signaling molecules for muscle differentiation. Carbonyl reductase 1 (CBR1) reduces highly reactive lipid aldehydes and catalyzes a variety of endogenous and xenobiotic carbonyl compounds. However, the role of CBR1 in muscle differentiation remains unclear. In this study, we found that CBR1 plays a crucial role in differentiation of muscle-derived C2C12 cells. Our results clearly show that CBR1 is upregulated at the transcript level during differentiation. Consistently, CBR1 was increased during skeletal muscle regeneration in tibialis anterior muscle after injury induced by cardiotoxin. The transcriptional upregulation of CBR1 was found to be controlled by nuclear factor erythroid 2-related factor 2 (Nrf2), and Nrf2 knockdown with specific siRNA inhibited muscle differentiation. Furthermore, intracellular ROS levels and lipid peroxidation were increased in cells transfected with CBR1 siRNA, or in cells treated with the selective CBR1 inhibitor, Hydroxy-PP-Me. Subsequently, the increased ROS levels diminished muscle cell differentiation. All together, we conclude that CBR1 plays a critical role in controlling redox balance and detoxifying lipid peroxidation during muscle differentiation and regeneration.

Age-Related Changes in Hepatic Activity and Expression of Detoxification Enzymes in Male Rats

Process of aging is accompanied by changes in the biotransformation of xenobiotics and impairment of normal cellular functions by free radicals. Therefore, this study was designed to determine age-related differences in the activities and/or expressions of selected drug-metabolizing and antioxidant enzymes in young and old rats. Specific activities of 8 drug-metabolizing enzymes and 4 antioxidant enzymes were assessed in hepatic subcellular fractions of 6-week-old and 21-month-old male Wistar rats. Protein expressions of carbonyl reductase 1 (CBR1) and glutathione S-transferase (GST) were determined using immunoblotting. Remarkable age-related decrease in specific activities of CYP2B, CYP3A, and UDP-glucuronosyl transferase was observed, whereas no changes in activities of CYP1A2, flavine monooxygenase, aldo-keto reductase 1C, and antioxidant enzymes with advancing age were found. On the other hand, specific activity of CBR1 and GST was 2.4 folds and 5.6 folds higher in the senescent rats compared with the young ones, respectively. Interindividual variability in CBR1 activity increased significantly with rising age. We suppose that elevated activities of GST and CBR1 may protect senescent rats against xenobiotic as well as eobiotic electrophiles and reactive carbonyls, but they may alter metabolism of drugs, which are CBR1 and especially GSTs substrates.

Identification of Carbonyl Reductase 1 as a Resveratrol-Binding Protein by Affinity Chromatography Using 4'-Amino-3,5-dihydroxy-trans-stilbene

The mechanisms by which resveratrol (3,4',5-trihydroxy-trans-stilbene) elicits diverse health benefits remain unclear because the intracellular target molecules of resveratrol are poorly defined. We screened resveratrol-binding proteins from lysates of MCF-7 breast cancer cells using resveratrol-affinity resin, which was constructed by immobilizing 4'-amino-3,5-dihydroxy-trans-stilbene on activated CH-Sepharose. On SDS-PAGE, two bands were detected as proteins that specifically bound to the resveratrol-affinity resin. One of these, a 30-kDa protein, was identified as human carbonyl reductase 1 (CBR1) by hybrid linear ion trap/time-of-flight mass spectrometry. Similarly, recombinant CBR1 bound to the resveratrol-affinity resin in the absence of resveratrol, but not in the presence of resveratrol. Among its activities, CBR1 catalyzes a NADPH-dependent reduction of the anticancer drug doxorubicin to the cardiotoxin doxorubicinol. The effects of doxorubicin on viability of MCF-7 cells were enhanced by resveratrol, 3,5-dihydroxy-4'-methoxy-trans-stilbene, 3,4'-dihydroxy-5-methoxy-trans-stilbene, and 4'-amino-3,5-dihydroxy-trans-stilbene at concentrations of 1 and 10 μM. Resveratrol and these derivatives inhibited CBR1 activities to a similar degree at concentrations of 100 and 200 μM. However, 3,5-dimethoxy-4'-hydroxy-trans-stilbene and m-hydroquinone had no influence on doxorubicin cytotoxicity or CBR1 activity. Resveratrol inhibited CBR1 activity through an apparent mix of competitive (Ki=55.8 μM) and noncompetitive (αKi=164 μM; α=2.98) inhibition kinetics. These results indicate that (i) resveratrol enhances the cytotoxic effects of doxorubicin on MCF-7 cells; (ii) the moiety that contains the 3,5-dihydroxyl groups of resveratrol, but not the m-hydroquinone structure alone, is required to bind CBR1; and (iii) resveratrol acts as a mixed-type inhibitor of CBR1 activity on doxorubicin.