Carbonyl Reductase 1 Inhibitor Research Articles

Loxoprofen enhances intestinal barrier function via generation of its active metabolite by carbonyl reductase 1 in differentiated Caco-2 cells

Nonsteroidal anti-inflammatory drugs (NSAIDs) are used worldwide as antipyretic analgesics and agents for rheumatoid arthritis and osteoarthritis, but known to cause damage to the gastrointestinal mucosae as their serious adverse effects. Few studies showed the impairment of intestinal epithelial barrier function (EBF) by high concentrations (0.5–1 mM) of NSAIDs, but the underlying mechanism is not fully understood. This study is aimed at clarifying effects at a low concentration (50 μM) of three NSAIDs, loxoprofen (Lox), ibuprofen and indomethacin, on intestinal EBF using human intestinal epithelial-like Caco-2 cells. Among those NSAIDs, Lox increased the transepithelial electric resistance (TER) value, decreased the paracellular Lucifer yellow CH (LYCH) permeability, and upregulated claudin (CLDN)-1, −3 and −5, indicating that low doses of Lox enhanced EBF through increasing expression of CLDNs. Lox is known to be metabolized to a pharmacologically active metabolite, (2S,1′R,2′S)-loxoprofen alcohol (Lox-RS), by carbonyl reductase 1 (CBR1), which is highly expressed in human intestine. CBR1 was expressed in the Caco-2 cells, and the pretreatment with a CBR1 inhibitor suppressed both the Lox-evoked CLDN upregulation and EBF enhancement. In addition, the treatment of the cells with Lox-RS resulted in higher TER value and lower LYCH permeability than those with Lox. Thus, Lox-RS synthesized by CBR1 may greatly contribute to the improving efficacy of Lox on the barrier function. Since EBF is decreased in inflammatory bowel disease, we finally examined the effect of Lox on EBF using the Caco-2/THP-1 co-culture system, which is used as an in vitro inflammatory bowel disease model. Lox significantly recovered EBF which was impaired by inflammatory cytokines secreted from THP-1 macrophages. These in vitro observations suggest that Lox enhances intestinal EBF, for which the metabolism of Lox to Lox-RS by CBR1 has an important role.

Synergistic anticancer activity of doxorubicin and piperlongumine on DU-145 prostate cancer cells – The involvement of carbonyl reductase 1 inhibition

One of the causes of therapeutic failure of chemotherapy is cancer cell resistance. In the case of anthracyclines, many resistance mechanisms have been described. One of them assumes the role of carbonyl reductase 1 (CBR1), a cytosolic enzyme that is responsible for the biotransformation process of anthracyclines to less active, undesirable metabolites. Therefore, CBR1 inhibitors are considered for use as a chemosensitizing agents. In the present study, piperlongumine (PL), a Piper longum L. alkaloid that has previously been described as a CBR1 inhibitor, was investigated for its chemosensitizing properties in co-treatment with doxorubicin (DOX). The biotransformation process of DOX in the presence of PL was tracked using human cytosol fraction and LC-MS, then a molecular modeling study was conducted to predict the interaction of PL with the active site of the CBR1. The biological interaction between DOX and PL was investigated using DU-145 prostate cancer cells. Cytotoxic and antiproliferative properties of DOX and PL were examined, and the type and potency of interaction was quantified by Combination Index. The mechanism of the cell death induced by the agents was investigated by flow cytometry and the anti-invasive properties of the drugs were determined by monitoring the movement of individual cells. PL showed dose-dependent inhibition of DOX metabolism in cytosol, which resulted in less doxorubicinol (DOXol) metabolite being formed. The possible mechanism of CBR1 inhibition was explained through molecular modeling studies by prediction of PL's binding mode in the active site of the enzyme's crystal structure-based model. DOX and PL showed a synergistic antiproliferative and proapoptotic effect on cancer cells. Significant anti-invasive properties of the combination of DOX and PL were found, but when the drugs were used separately they did not alter the cancer cells' motility. Cell motility inhibition was accompanied by significant changes in cytoskeleton architecture. DOX and PL used in co-treatment showed significant synergistic anticancer properties. Inhibition of DOX metabolism by PL was found to be a mechanism that was likely to be responsible for the observed interaction.

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.

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).

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.

Carbonyl Reductase 1 Offers a Novel Therapeutic Target to Enhance Leukemia Treatment by Arsenic Trioxide

Arsenic trioxide (As2O3) is used, in current practice, as an effective chemotherapeutic agent for acute promyelocytic leukemia (APL). However, the side effects and relatively low efficacy of As2O3 in treating other leukemias have limited its wider use in therapeutic applications. In the present study, we found that the expression of carbonyl reductase 1 (CBR1) affects the resistance to As2O3 in leukemias, including APL; As2O3 upregulated CBR1 expression at the transcriptional level by stimulating the activity of the transcription factor activator protein-1. Moreover, CBR1 overexpression was sufficient to protect cells against As2O3 through modulation of the generation of reactive oxygen species, whereas the attenuation of CBR1 was sufficient to sensitize cells to As2O3. A combination treatment with the specific CBR1 inhibitor hydroxy-PP-Me remarkably increased As2O3-induced apoptotic cell death compared with As2O3 alone, both in vitro and in vivo. These results were confirmed in primary cultured human acute and chronic myeloid leukemia cells, with no significant cell death observed in normal leukocytes. Taken together, our findings indicate that CBR1 contributes to the low efficacy of As2O3 and, therefore, is a rational target for the development of combination chemotherapy with As2O3 in diverse leukemias including APL.

Sex differences in improved efficacy of doxorubicin chemotherapy in Cbr1+/− mice

The anthracycline chemotherapeutic agent doxorubicin is converted by the enzyme carbonyl reductase 1 (CBR1) into its cardiotoxic metabolite doxorubicinol. Cbr1+/- mice have been shown to be protected from doxorubicin-induced cardiotoxicity, and the inhibition of CBR1 activity may be a useful means of ameliorating the side effects of doxorubicin in patients undergoing chemotherapy. Because reduced conversion to doxorubicinol increases circulating levels of the more effective parent drug doxorubicin, it was hypothesized that therapeutic efficacy against tumors might also be enhanced. Cbr1+/- mice were bred to mice transgenic for the polyomavirus middle T antigen (PyVT) to create offspring with palpable mammary tumors. Latency to initial tumor formation was similar in Cbr1+/- and Cbr1+/+ animals. Tumor regression was improved in Cbr1+/- animals, but only in male mice. Western blotting showed a marked sex difference in protein levels, with a much higher expression of Cbr1 in the female kidney and liver. Thus, the combined effects of a naturally low expression and the heterozygous Cbr1 null allele seem to have enhanced tumor regression in Cbr1+/- males. Future efforts to design a clinical CBR1 inhibitor to protect patients from the cardiac side effects of doxorubicin treatment should evaluate the effect of sex on anticancer efficacy.

Human carbonyl reductase 1 upregulated by hypoxia renders resistance to apoptosis in hepatocellular carcinoma cells

Human carbonyl reductase1 (CBR1) has been reported to protect cells against lipid peroxidation. Since human hepatocellular carcinoma (HCC) cells are under oxidative stress in hypoxic conditions, we tested if CBR1 is upregulated by hypoxia inducible factor (HIF)-1α, helps tumor growth under hypoxia, and renders chemoresistance to cisplatin and doxorubicin in HCC. Luciferase, EMSA, and chromatin immunoprecipitation (ChIP) assays were performed to analyze whether HIF-1α transactivates CBR1 promoter. CBR1 overexpression, siRNA, and inhibitors were used to study the role of CBR1 in tumor survival under hypoxia and chemoresistance to cisplatin and doxorubicin in HCC. FACS and Western blot analysis for oxidative stress markers were performed to measure ROS. Immunohistochemistry (IHC) was performed to analyze CBR1 expression in 78 cases of HCC and 123 cases of colon cancer tissues. The CBR1 promoter was activated by HIF-1α. CBR1 overexpression enhanced cell survival by decreasing oxidative stress under hypoxia, cisplatin, and doxorubicin treatment. CBR1-siRNA increased apoptosis via increasing oxidative stress. Combinational therapy of CBR1 inhibitors with cisplatin or doxorubicin enhanced cell death in HCC cells. IHC showed CBR1 overexpression in 56 (72%) out of 78 HCC and 88 (72%) out of 123 colon cancer cases. Overexpressed CBR1 by HIF-1α plays important roles in tumor growth under hypoxia and chemoresistance to anticancer drugs. The inhibition of CBR1 by specific inhibitors enhances anticancer drug efficacy in HCC. Therefore, we concluded that CBR1 is a good molecular target for the development of anticancer drugs for HCC patients.

Inhibition of Polymorphic Human Carbonyl Reductase 1 (CBR1) by the Cardioprotectant Flavonoid 7-monohydroxyethyl Rutoside (monoHER)

Carbonyl reductase 1 (CBR1) reduces the anticancer anthracyclines doxorubicin and daunorubicin into the cardiotoxic metabolites doxorubicinol and daunorubicinol. We evaluated whether the cardioprotectant monoHER inhibits the activity of polymorphic CBR1. We performed enzyme kinetic studies with monoHER, CBR1 (CBR1 V88 and CBR1 I88) and anthracycline substrates. We also characterized CBR1 inhibition by the related flavonoids triHER and quercetin. MonoHER inhibited the activity of CBR1 V88 and CBR1 I88 in a concentration-dependent manner. The IC(50) values of monoHER were lower for CBR1 I88 compared to CBR1 V88 for the substrates daunorubicin and doxorubicin (daunorubicin, IC(50)-CBR1 I88 = 164 microM vs. IC(50)-CBR1 V88 = 219 microM; doxorubicin, IC(50)-CBR1 I88 = 37 microM vs. IC(50)-CBR1 V88 = 59 microM; p < 0.001). Similarly, the flavonoids triHER and quercetin exhibited lower IC(50) values for CBR1 I88 compared to CBR1 V88 (p < 0.001). MonoHER acted as a competitive CBR1 inhibitor when using daunorubicin as a substrate Ki = 45 +/- 18 microM. MonoHER acted as an uncompetitive CBR1 inhibitor for the small quinone substrate menadione Ki = 33 +/- 17 microM. The cardioprotectant monoHER inhibits CBR1 activity. CBR1 V88I genotype status and the type of anthracycline substrate dictate the inhibition of CBR1 activity.

An unbiased cell morphology-based screen for new, biologically active small molecules.

We have implemented an unbiased cell morphology–based screen to identify small-molecule modulators of cellular processes using the Cytometrix (TM) automated imaging and analysis system. This assay format provides unbiased analysis of morphological effects induced by small molecules by capturing phenotypic readouts of most known classes of pharmacological agents and has the potential to read out pathways for which little is known. Four human-cancer cell lines and one noncancerous primary cell type were treated with 107 small molecules comprising four different protein kinase–inhibitor scaffolds. Cellular phenotypes induced by each compound were quantified by multivariate statistical analysis of the morphology, staining intensity, and spatial attributes of the cellular nuclei, microtubules, and Golgi compartments. Principal component analysis was used to identify inhibitors of cellular components not targeted by known protein kinase inhibitors. Here we focus on a hydroxyl-substituted analog (hydroxy-PP) of the known Src-family kinase inhibitor PP2 because it induced cell-specific morphological features distinct from all known kinase inhibitors in the collection. We used affinity purification to identify a target of hydroxy-PP, carbonyl reductase 1 (CBR1), a short-chain dehydrogenase-reductase. We solved the X-ray crystal structure of the CBR1/hydroxy-PP complex to 1.24 Å resolution. Structure-based design of more potent and selective CBR1 inhibitors provided probes for analyzing the biological function of CBR1 in A549 cells. These studies revealed a previously unknown function for CBR1 in serum-withdrawal-induced apoptosis. Further studies indicate CBR1 inhibitors may enhance the effectiveness of anticancer anthracyclines. Morphology-based screening of diverse cancer cell types has provided a method for discovering potent new small-molecule probes for cell biological studies and anticancer drug candidates.