Aldo-keto Reductase 1C3 Research Articles

Exploring the Potential of Sulfur Moieties in Compounds Inhibiting Steroidogenesis.

This study reports on the synthesis and evaluation of novel compounds replacing the nitrogen-containing heterocyclic ring on the chemical backbone structure of cytochrome P450 17α-hydroxylase/12,20-lyase (CYP17A1) inhibitors with a phenyl bearing a sulfur-based substituent. Initial screening revealed compounds with marked inhibition of CYP17A1 activity. The selectivity of compounds was thereafter determined against cytochrome P450 21-hydroxylase, cytochrome P450 3A4, and cytochrome P450 oxidoreductase. Additionally, the compounds showed weak inhibitory activity against aldo-keto reductase 1C3 (AKR1C3). The compounds' impact on steroid hormone levels was also assessed, with some notable modulatory effects observed. This work paves the way for developing more potent dual inhibitors specifically targeting CYP17A1 and AKR1C3.

In silico study of novel alpha tocopheroids as effective inhibitors of aldo-keto reductase 1c3 (AKR1C3) enzyme

Aldo-keto reductase 1C3 (AKR1C3) is a monomeric enzyme expressed in steroidogenic tissues such as the testis, prostate, uterus, and breast. Overexpression of this AKR1C3 is associated with vast cancers such as breast, colon, colorectal, endometrial, prostate, and acute myeloid leukaemia. Regarding the treatment of castration-resistant prostate cancer, breast cancer, and acute myeloid leukaemia, AKR1C3 inhibitors may offer clear advantages over currently available therapies. Thus, discovering novel and specific AKR1C3 inhibitors is a promising way to obstruct drug resistance in cancer. Derivatives of alpha-tocopherol and alpha-tocopheroids were selected as possible therapeutics to act as AKR1C3 inhibitors. The precise targets of several ligands were determined using computational screening methods. The molecular structure of AKR1C3 and its ligands were used as the foundation for in silico predictions, modelling, and dynamic simulations. Compounds were selected based on their biological properties and filtered according to their ADMET and drug-likeness properties. Additionally, simulations of all-atom molecular dynamics on AKR1C3 with the cleared compounds revealed stability over the simulated trajectories of 100 ns. When seen collectively, alpha-tocospiro A may be considered prospective AKR1C3 inhibitors for creating anticancer therapies. Communicated by Ramaswamy H. Sarma

Aldo-Keto Reductase 1C3 Inhibitor Prodrug Improves Pharmacokinetic Profile and Demonstrates In Vivo Efficacy in a Prostate Cancer Xenograft Model.

Aldo-keto reductase 1C3 (AKR1C3) is overexpressed in castration-resistant prostate cancer where it acts to drive proliferation and aggressiveness by producing androgens. The reductive action of the enzyme leads to chemoresistance development against various clinical antineoplastics across a range of cancers. Herein, we report the continued optimization of selective AKR1C3 inhibitors and the identification of 5r, a potent AKR1C3 inhibitor (IC50 = 51 nM) with >1216-fold selectivity for AKR1C3 over closely related isoforms. Due to the cognizance of the poor pharmacokinetics associated with free carboxylic acids, a methyl ester prodrug strategy was pursued. The prodrug 4r was converted to free acid 5r in vitro in mouse plasma and in vivo. The in vivo pharmacokinetic evaluation revealed an increase in systemic exposure and increased the maximum 5r concentration compared to direct administration of the free acid. The prodrug 4r demonstrated a dose-dependent effect to reduce the tumor volume of 22Rv1 prostate cancer xenografts without observed toxicity.

Development of Biaryl-Containing Aldo-Keto Reductase 1C3 (AKR1C3) Inhibitors for Reversing AKR1C3-Mediated Drug Resistance in Cancer Treatment.

Aldo-keto reductase 1C3 (AKR1C3) is correlated with tumor development and chemotherapy resistance. The catalytic activity of the enzyme has been recognized as one of the important factors in inducing anthracycline (ANT) resistance in cancer cells. Inhibition of AKR1C3 activity may provide a promising approach to restore the chemosensitivity of ANT-resistant cancers. Herein, a series of biaryl-containing AKR1C3 inhibitors has been developed. The best analogue S07-1066 selectively blocked AKR1C3-mediated reduction of doxorubicin (DOX) in MCF-7 transfected cell models. Furthermore, co-treatment of S07-1066 significantly synergized DOX cytotoxicity and reversed the DOX resistance in MCF-7 cells overexpressing AKR1C3. The potential synergism of S07-1066 over DOX cytotoxicity was demonstrated in vitro and in vivo. Our findings indicate that inhibition of AKR1C3 potentially enhances the therapeutic efficacy of ANTs and even suggests that AKR1C3 inhibitors may serve as effective adjuvants to overcome AKR1C3-mediated chemotherapy resistance in cancer treatment.

In vitro evaluation of the reductase activities of human AKR1C3 allelic variants.

Aldo-keto reductase 1C3 (AKR1C3) plays a role in the detoxification and activation of clinical drugs by catalyzing reduction reactions. There are approximately 400 single nucleotide polymorphisms in the AKR1C3 gene, but their impact on the enzyme activity is still unclear. This study aimed to clarify the effects of single nucleotide polymorphisms of AKR1C3 with more than 0.5% global minor allele frequency on the reductase activities for its typical substrates. Recombinant AKR1C3 wild-type and R66Q, E77G, C145Y, P180S, or R258C variants were constructed using insect Sf21 cells, and reductase activities for acetohexamide, doxorubicin, and loxoprofen by recombinant AKR1C3s were measured by LC‒MS/MS. Among the variants tested, the C145Y variant showed remarkably low (6-14% of wild-type) intrinsic clearances of reductase activities for all three drugs. Reductase activities of these three drugs were measured using 34 individual Japanese liver cytosols, revealing that heterozygotes of the SNP g.55101G>A tended to show lower reductase activities for three drugs than homozygotes of the wild-type. Furthermore, genotyping of the SNP g.55101G>A causing C145Y in 96 Caucasians, 166 African Americans, 192 Koreans, and 183 Japanese individuals was performed by polymerase chain reaction-restriction fragment length polymorphism. This allelic variant was specifically detected in Asians with allele frequencies of 6.8% and 3.6% in Koreans and Japanese, respectively. To conclude, an AKR1C3 allele with the SNP g.55101G>A causing C145Y would be one of the causal factors for interindividual variabilities in the efficacy and toxicity of drugs reduced by AKR1C3. Significance Statement This is the first study to clarify that the AKR1C3 allele with the SNP g.55101G>A causing C145Y results in a decrease in reductase activity. Since the allele was specifically observed in Asians, the allele would be a factor causing an interindividual variability in sensitivity of drug efficacy or toxicity of drugs reduced by AKR1C3 in Asians.

Interplay of Cortisol, Testosterone, and Abdominal Fat Mass in Normal-weight Women With Polycystic Ovary Syndrome.

Ovarian and adrenal steroidogenesis underlie endocrine-metabolic dysfunction in polycystic ovary syndrome (PCOS). Adipocytes express aldo-keto reductase 1C3 and type 1 11β-hydroxysteroid dehydrogenase, which modulate peripheral androgen and cortisol production. To compare serum adrenal steroids, including 11-oxygenated androgens (11-oxyandrogens), cortisol, and cortisone between normal-weight women with PCOS and body mass index- and age-matched ovulatory women with normal-androgenic profiles (controls), and assess whether adrenal steroids associate with abdominal adipose deposition. Prospective, cross-sectional, cohort study. Academic medical center. Twenty normal-weight women with PCOS and 20 body mass index-/age-matched controls. Blood sampling, IV glucose tolerance testing, and total-body dual-energy x-ray absorptiometry. Clinical characteristics, hormonal concentrations, and body fat distribution. Women with PCOS had higher serum total/free testosterone (T) and androstenedione (A4) levels and a greater android/gynoid fat mass than controls (androgens P < .001; android/gynoid fat mass ratio, P = .026). Serum total/free T and A4 levels correlated positively with android/gynoid fat mass ratio in all women combined (P < .025, all values). Serum 11ß-hydroxyA4, 11-ketoA4, 11ß-hydroxyT, 11-ketoT, cortisol, and cortisone levels were comparable between female types and unrelated to body fat distribution. Serum 11-oxyandrogens correlated negatively with % total body fat, but lost significance adjusting for cortisol. Serum cortisol levels, however, correlated inversely with android fat mass (P = .021), with a trend toward reduced serum cortisol to cortisone ratio in women with PCOS vs controls (P = .075), suggesting diminished 11β-hydroxysteroid dehydrogenase activity. Reduced cortisol may protect against preferential abdominal fat mass in normal-weight PCOS women with normal serum 11-oxyandrogens.

In Silico and In Vitro Assessment of Carbonyl Reductase 1 Inhibition Using ASP9521-A Potent Aldo-Keto Reductase 1C3 Inhibitor with the Potential to Support Anticancer Therapy Using Anthracycline Antibiotics.

Anthracycline antibiotics (ANT) are among the most widely used anticancer drugs. Unfortunately, their use is limited due to the development of drug resistance and cardiotoxicity. ANT metabolism, performed mainly by two enzymes-aldo-keto reductase 1C3 (AKR1C3) and carbonyl reductase 1 (CBR1)-is one of the proposed mechanisms generated by the described effects. In this study, we evaluated the CBR1 inhibitory properties of ASP9521, a compound already known as potent AKR1C3 inhibitor. First, we assessed the possibility of ASP9521 binding to the CBR1 catalytic site using molecular docking and molecular dynamics. The research revealed a potential binding mode of ASP9521. Moderate inhibitory activity against CBR1 was observed in studies with recombinant enzymes. Finally, we examined whether ASP9521 can improve the cytotoxic activity of daunorubicin against human lung carcinoma cell line A549 and assessed the cardioprotective properties of ASP9521 in a rat cardiomyocytes model (H9c2) against doxorubicin- and daunorubicin-induced toxicity. The addition of ASP9521 ameliorated the cytotoxic activity of daunorubicin and protected rat cardiomyocytes from the cytotoxic effect of both applied drugs. Considering the favorable bioavailability and safety profile of ASP9521, the obtained results encourage further research. Inhibition of both AKR1C3 and CBR1 may be a promising method of overcoming ANT resistance and cardiotoxicity.

Description of Chemical Synthesis, Nuclear Magnetic Resonance Characterization and Biological Activity of Estrane-Based Inhibitors/Activators of Steroidogenesis.

Steroid hormones play a crucial role in several aspects of human life, and steroidogenesis is the process by which hormones are produced from cholesterol using several enzymes that work in concert to obtain the appropriate levels of each hormone at the right time. Unfortunately, many diseases, such as cancer, endometriosis, and osteoporosis as examples, are caused by an increase in the production of certain hormones. For these diseases, the use of an inhibitor to block the activity of an enzyme and, in doing so, the production of a key hormone is a proven therapeutic strategy whose development continues. This account-type article focuses on seven inhibitors (compounds 1-7) and an activator (compound 8) of six enzymes involved in steroidogenesis, namely steroid sulfatase, aldo-keto reductase 1C3, types 1, 2, 3, and 12 of the 17β-hydroxysteroid dehydrogenases. For these steroid derivatives, three topics will be addressed: (1) Their chemical synthesis from the same starting material, estrone, (2) their structural characterization using nuclear magnetic resonance, and (3) their in vitro or in vivo biological activities. These bioactive molecules constitute potential therapeutic or mechanistic tools that could be used to better understand the role of certain hormones in steroidogenesis.

MP20-16 NOVEL FUNCTION OF PTUPB IN AKR1C3/AR/AR-V7 AXIS INHIBITION AND ENZALUTAMIDE SENSITIZATION IN ADVANCED PROSTATE CANCER

You have accessJournal of UrologyCME1 Apr 2023MP20-16 NOVEL FUNCTION OF PTUPB IN AKR1C3/AR/AR-V7 AXIS INHIBITION AND ENZALUTAMIDE SENSITIZATION IN ADVANCED PROSTATE CANCER Joy Yang, Pengfei Xu, Bo Chen, Shu Ning, Hans Adomat, Sung-Hee Hwang, Allen Gao, Martin Gleave, Bruce Hammock, Christopher Evans, and Chengfei Liu Joy YangJoy Yang More articles by this author , Pengfei XuPengfei Xu More articles by this author , Bo ChenBo Chen More articles by this author , Shu NingShu Ning More articles by this author , Hans AdomatHans Adomat More articles by this author , Sung-Hee HwangSung-Hee Hwang More articles by this author , Allen GaoAllen Gao More articles by this author , Martin GleaveMartin Gleave More articles by this author , Bruce HammockBruce Hammock More articles by this author , Christopher EvansChristopher Evans More articles by this author , and Chengfei LiuChengfei Liu More articles by this author View All Author Informationhttps://doi.org/10.1097/JU.0000000000003245.16AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookLinked InTwitterEmail Abstract INTRODUCTION AND OBJECTIVE: The steroidogenic enzyme Aldo-keto Reductase 1C3 (AKR1C3) is a major factor that contributes to the progression of castration-resistant prostate cancer (CRPC) and treatment failure. However, none of the available AKR1C3 inhibitors has shown promising results in clinical settings. Thus, there is an unmet need to develop superior AKR1C3 inhibitors for clinical testing. METHODS: PTUPB was synthesized by linking two pharmacophores, celecoxib and TPPU. In vitro and in vivo pharmacological activities of PTUPB in combination with enzalutamide were evaluated in several enzalutamide-resistant cell lines, conditional reprogrammed cell cultures, and patient-derived organoid models. The mechanism of action of PTUPB was examined via RNA sequence analysis, immunoblot analysis, co-immunoprecipitation, steroid measurement, and enzyme activity assays. Animal studies using PTUPB in combination with enzalutamide were performed in the castration-relapse VCaP tumor model. RESULTS: AKR1C3 levels are reciprocally regulated by the full-length androgen receptor (AR-FL) by binding to the distal enhancer region of AKR1C3. A novel function of PTUPB in AKR1C3 inhibition was discovered: at the same dose (0.5 µM) PTUPB suppressed the AKR1C3 activity more than indomethacin (60% vs. 40%) and celeboxib (60% vs. 0, p<0.05). And at the same dose (20 µM), PTUPB was more effective than indomethacin (98% vs. 22% of inhibition, p<0.05) and celecoxib (98% vs. 60% inhibition, p<0.05) in inhibiting CRPC cell growth. PTUPB synergizes with enzalutamide treatment for tumor suppression and gene signature regulation. Combination treatment with PTUPB and enzalutamide provides benefits by blocking AR and its variant (AR-V7) signaling, which inhibits the growth of VCaP castration-relapse tumors with 2.8-fold more inhibition (p<0.05) in tumor weights between the single (PTUPB) and combination treatments (Enza+PTUPB). There was also more inhibition in the growth of the patient-derived xenograft organoids by PTUPB alone or in combination with Enza. CONCLUSIONS: Targeting the ARK1C3/AR/AR-V7 axis with PTUPB and enzalutamide may overcome drug resistance to AR signaling inhibitors in advanced prostate cancer. Source of Funding: This work was supported in part by grants NIH/NCI R37CA249108 (C. Liu) and R01CA251253 (C. Liu) © 2023 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 209Issue Supplement 4April 2023Page: e281 Advertisement Copyright & Permissions© 2023 by American Urological Association Education and Research, Inc.MetricsAuthor Information Joy Yang More articles by this author Pengfei Xu More articles by this author Bo Chen More articles by this author Shu Ning More articles by this author Hans Adomat More articles by this author Sung-Hee Hwang More articles by this author Allen Gao More articles by this author Martin Gleave More articles by this author Bruce Hammock More articles by this author Christopher Evans More articles by this author Chengfei Liu More articles by this author Expand All Advertisement PDF downloadLoading ...

A novel AKR1C3 specific prodrug AST-3424 and its combination therapy in hepatocellular carcinoma

ObjectiveAST-3424 is a novel specific aldo-keto reductase 1C3 (AKR1C3) prodrug that releases a DNA alkylating reagent upon reduction by AKR1C3. This study aimed to evaluate the efficacy and safety of AST-3424 in patient-derived tumor xenograft (PDTX) model and orthotopic model against hepatocellular carcinoma (HCC). Materials and methodPDTX models derived from three HCC patients and orthotopic mice models using HepG2 cells were developed. The mice were treated with AST-3424 alone or combined with other drugs (oxaliplatin, apatinib, sorafenib and elemene in PDTX models, oxaliplatin and 5- fluorouracil in orthotopic models). The tumor volume and weight, as well as the mice weight were assessed. The liver tumor and transplanted tumor were removed for histological, immunohistochemical and Western blot detection in orthotopic model experiments. ResultsAST-3424 could inhibit tumor growth in HCC PDTX models and orthotopic models, with no difference in safety compared with other marketed drugs, and the drug combination did not increase toxicity. The inhibitory effect of combination treatment was more obvious than which used alone. The reduction of AKR1C3 expression was negatively correlated with AST-3424 dose. ConclusionAST-3424 had a promising effect against HCC in PDTX model and orthotopic model with good safety. It could promote the sensitivity of other drugs without increasing toxicity. Clinical trials are warranted to further certify its antitumor effect and safety.

Influence of aldo–keto reductase 1C3 polymorphisms in early-onset female psoriasis patients

The principal pathology of psoriasis is impaired skin barrier function, epidermal thickening, and granular layer loss. Exposure to extrinsic factors such as tobacco smoke and air pollutants is associated with the development of psoriasis. Aryl hydrocarbon receptors (AHRs) are activated by extrinsic factors associated with the development of psoriasis and act as transcriptional regulators. Expression of aldo–keto reductase (AKR) 1C3 in the epidermal spinous layer regulates epidermal keratinocyte differentiation via the AHR signaling pathway. We investigated whether single nucleotide polymorphisms (SNPs) in AKR1C3 are associated with the pathogenesis of psoriasis. The proportions of rs12529 G/C, C/C variants, and rs12387 A/A, A/G variants were twofold higher in Japanese psoriasis patients (n = 231) compared with a Japanese healthy cohort. The SNPs were significantly more common than the majority variants in female patients with disease onset ≤ 22 years of age. Patients with rs12529 G > C and rs12387 A > G SNPs exhibited significantly lower AKR1C3 expression and higher expression of late differentiation markers. In conclusion, AKR1C3 downregulation caused by rs12529 G > C and rs12387 A > G SNPs in the epidermis induces abnormal early differentiation of keratinocytes and skin barrier dysfunction, which may contribute to the genetic pathogenesis of psoriasis in young females.

Role of Human Aldo-Keto Reductases and Nuclear Factor Erythroid 2-Related Factor 2 in the Metabolic Activation of 1-Nitropyrene via Nitroreduction in Human Lung Cells.

1-Nitropyrene (1-NP) is a constituent of diesel exhaust and classified as a group 2A probable human carcinogen. The metabolic activation of 1-NP by nitroreduction generates electrophiles that can covalently bind DNA to form mutations to contribute to cancer causation. NADPH-dependent P450 oxidoreductase (POR), xanthine oxidase (XO), aldehyde oxidase (AOX), and NAD(P)H/quinone oxidoreductase 1 (NQO1) may catalyze 1-NP nitroreduction. We recently found that human recombinant aldo-keto reductases (AKRs) 1C1-1C3 catalyze 1-NP nitroreduction. NQO1 and AKR1C1-1C3 are genes induced by nuclear factor erythroid 2-related factor 2 (NRF2). Despite this knowledge, the relative importance of these enzymes and NRF2 to 1-NP nitroreduction is unknown. We used a combination of pharmacological and genetic approaches to assess the relative importance of these enzymes and NRF2 in the aerobic nitroreduction of 1-NP in human bronchial epithelial cells, A549 and HBEC3-KT. 1-NP nitroreduction was assessed by the measurement of 1-aminopyrene (1-AP), the six-electron reduced metabolite of 1-NP, based on its intrinsic fluorescence properties (λex and λem). We found that co-treatment of 1-NP with salicylic acid, an AKR1C1 inhibitor, or ursodeoxycholate, an AKR1C2 inhibitor, for 48 h decreased 1-AP production relative to 1-NP treatment alone (control) in both cell lines. R-Sulforaphane or 1-(2-cyano-3,12,28-trioxooleana-1,9(11)-dien-28-yl)-1H-imidazole (CDDO-Im), two NRF2 activators, each increased 1-AP production relative to control only in HBEC3-KT cells, which have inducible NRF2. Inhibitors of POR, NQO1, and XO failed to modify 1-AP production relative to control in both cell lines. Importantly, A549 wild-type cells with constitutively active NRF2 produced more 1-AP than A549 cells with heterozygous expression of NFE2L2/NRF2, which were able to produce more 1-AP than A549 cells with homozygous knockout of NFE2L2/NRF2. Together, these data show dependence of 1-NP metabolic activation on AKR1Cs and NRF2 in human lung cells. This is the second example whereby NFE2L2/NRF2 is implicated in the carcinogenicity of diesel exhaust constituents.

X-ray structure of human aldo-keto reductase 1C3 in complex with a bile acid fused tetrazole inhibitor: experimental validation, molecular docking and structural analysis.

Aldo-keto reductase 1C3 (AKR1C3) catalyzes the reduction of androstenedione to testosterone and reduces the effectiveness of chemotherapeutics. AKR1C3 is a target for treatment of breast and prostate cancer and AKR1C3 inhibition could be an effective adjuvant therapy in the context of leukemia and other cancers. In the present study, steroidal bile acid fused tetrazoles were screened for their ability to inhibit AKR1C3. Four C24 bile acids with C-ring fused tetrazoles were moderate to strong AKR1C3 inhibitors (37-88% inhibition), while B-ring fused tetrazoles had no effect on AKR1C3 activity. Based on a fluorescence assay in yeast cells, these four compounds displayed no affinity for estrogen receptor-α, or the androgen receptor, suggesting a lack of estrogenic or androgenic effects. A top inhibitor showed specificity for AKR1C3 over AKR1C2, and inhibited AKR1C3 with an IC50 of ∼7 μM. The structure of AKR1C3·NADP+ in complex with this C-ring fused bile acid tetrazole was determined by X-ray crystallography at 1.4 Å resolution, revealing that the C24 carboxylate is anchored to the catalytic oxyanion site (H117, Y55); meanwhile the tetrazole interacts with a tryptophan (W227) important for steroid recognition. Molecular docking predicts that all four top AKR1C3 inhibitors bind with nearly identical geometry, suggesting that C-ring bile acid fused tetrazoles represent a new class of AKR1C3 inhibitors.

Detection and quantification of UV-transparent keto-androgens by dinitrophenylhydrazine derivatization for discontinuous kinetic assays.

Kinetic assays with recombinant enzymes are critical to determine the steady state kinetic parameters for androgen conversion to understand their role in androgen biosynthesis and metabolism. Detection and quantification of 5α-reduced androgens remain difficult to assay because they are UV-transparent compounds. Therefore, radioactive isotopic versions of these compounds are often required to conduct steady-state kinetics. Here we developed a derivatization protocol with dinitrophenylhydrazine (DNPH) to form hydrazones on the ketones of androgens enabling them to be detected by UV-reverse phase high performance liquid chromatography (RP-HPLC). We determined the kinetic parameters for the conversion of 5α-androstane-3,17-dione (5AD) to 5α-dihydrotestosterone (DHT), 11-keto-5α-androstane-3,17-dione (11K-5AD) to 11-keto-5α-dihydrotestosterone (11K-DHT), and 11β-hydroxy-5α-androstane-3,17-dione (11β-OH-5AD) to 11β-hydroxy-5α-dihydrotestosterone (11β-OH-DHT) catalyzed by recombinant aldo-keto reductase 1C3 (AKR1C3) as measured by product formation post DNPH derivatization.

Development of highly potent and specific AKR1C3 inhibitors to restore the chemosensitivity of drug-resistant breast cancer

Aldo-keto reductase 1C3 (AKR1C3) is overexpressed in multiple hormone related cancers, such as breast and prostate cancer, and is correlated with tumor development and aggressiveness. As a phase I biotransformation enzyme, AKR1C3 catalyzes the metabolic processes that lead to resistance to anthracyclines, the “gold standard” for breast cancer treatment. Novel approaches to restore the chemotherapy sensitivity of breast cancer are urgently required. Herein, we developed a new class of AKR1C3 inhibitors that demonstrated potent inhibitory activity and exquisite selectivity for closely related isoforms. The best derivative 27 (S19–1035) exhibits an IC50 value of 3.04 nM for AKR1C3 and >3289-fold selectivity over other isoforms. We determined the co-crystal structures of AKR1C3 with three of the inhibitors, providing a solid foundation for further structure-based drug optimization. Co-administration of these AKR1C3 inhibitors significantly reversed the doxorubicin (DOX) resistance in a resistant breast cancer cell line. Therefore, the novel AKR1C3 specific inhibitors developed in this work may serve as effective adjuvants to overcome DOX resistance in breast cancer treatment.

Availability of aldo-keto reductase 1C3 and ATP-binding cassette B1 as therapeutic targets for alleviating paclitaxel resistance in breast cancer MCF7 cells.

Paclitaxel (PTX) is frequently utilized for the chemotherapy of breast cancer, but its continuous treatment provokes hyposensitivity. Here, we established a PTX-resistant variant of human breast cancer MCF7 cells and found that acquiring the chemoresistance elicits a remarkable up-regulation of aldo-keto reductase (AKR) 1C3. MCF7 cell sensitivity to PTX toxicity was increased by pretreatment with AKR1C3 inhibitor and knockdown of this enzyme, and decreased by its overexpression, inferring a crucial role of AKR1C3 in the development of PTX resistance. The PTX-resistant cells were much less sensitive to 4-hydroxy-2-nonenal and acrolein, cytotoxic reactive aldehydes derived from ROS-mediated lipid peroxidation, compared with the parental cells. Additionally, the resistant cells lowered levels of 4-hydroxy-2-nonenal formed during PTX treatment, which was mitigated by pretreating with AKR1C3 inhibitor, suggesting that AKR1C3 procures the chemoresistance through facilitating the metabolism of the cytotoxic aldehyde. The gain of PTX resistance additively promoted the aberrant expression of an ATP-binding cassette (ABC) transporter ABCB1 among the ABC transporter isoforms. The combined treatment with AKR1C3 and ABCB1 inhibitors overcame the PTX resistance and cross-resistance to another taxane-based drug docetaxel. Collectively, combined treatment with AKR1C3 and ABCB1 inhibitors may exert an overcoming effect of PTX resistance in breast cancer.

ODP440 Characterization of the Major Single Nucleotide Polymorphic Variants of Aldo-Keto Reductase 1c3 (Type 5 17β-Hydroxysteroid Dehydrogenase)

Abstract Background Aldo-keto-reductase (AKR) 1C3, also known as type 517β-hydroxysteroid dehydrogenase and prostaglandin F synthase,is a member of the AKR superfamily that reduces aldehydes and ketones to primary and secondary alcohols. It plays an essential role in the peripheral formation of androgens and is associated with several steroid hormone dependent diseases including prostate cancer (PC), breast cancer, and polycystic ovarian syndrome (PCOS). Furthermore, AKR1C3 is implicated in castration resistant prostate cancer (CRPC) drug resistance on the basis that it is upregulated in CRPC tumors and metastases and overexpressed in cell culture &amp; xenograft models of Abiraterone and Enzalutamide resistance, where resistance is surmounted by AKR1C3 knockdown or treatment with the AKR1C3 inhibitor indomethacin. AKR1C3 has 14 nonsynonymous single nucleotide polymorphisms (nsSNPs) with different global frequencies and ethnic distributions. Association studies support their role in PC and other diseases, but detailed functional genomic studies of these variants are lacking. One study examined5 AKR1C3 nsSNPs for their ability to reduce exemestane, an aromatase inhibitor used to treat breast cancer, to 17β-dihydroexemestane, and reported a 17-250-fold reduction in catalytic efficiency of H5Q, E77G, K104D, and R258C variants compared to WT. This observation provided the impetus to examine impact of these variants on AKR1C3 function in the context of PC. We purified AKR1C3 WT, and the top four most frequently occurring nsSNPs, H5Q, E77G, K104D, and R258C, from E. coli to expand upon their characterization and illuminate functional differences that could affect disease outcome and treatment. METHODS: Kinetic measurements were made using either a continuous spectrophotometric assay monitoring the appearance of the reduced cofactor NADH, or a discontinuous RP UV-HPLC assay based on chromatographic separation of substrate and product. Inhibitor sensitivity studies were conducted with a fluorescence-based plate-reader assay monitoring enzyme activity via oxidation of a standard substrate in the presence of increasing inhibitor. Stability was assessed by monitoring the percent of maximum activity remaining via oxidation of a standard substrate over a range of increasing temperatures for each variant. Results We report no major deviations in kinetics in the top four most frequently occurring AKR1C3 variants compared to WT. Additionally, variants did not display differences in inhibitor sensitivity to competitive inhibitor indomethacin. However, the K104D variant lost activity at a lower temperature than WT, suggesting that this mutation results in a decrease in stability. Conclusions Altogether, these findings contrast with previous claims that AKR1C3 variants, mainly H5Q, give rise to significant advantage or disadvantage over WT in PC and other diseases, and suggests that patients with AKR1C3 dependent diseases would likely be overall unaffected by variant status. Presentation: No date and time listed

ODP001 AKR1C2 Activity in Abdominal Adipose Tissue of Women with or without Excess Visceral Adiposity

Abstract Background Adipose tissue is known to play an active role in androgen turnover, as illustrated by the presence of enzymes of the aldo-keto reductase 1C family such as AKR1C2 and AKR1C3, respectively coding for 3α-hydroxysteroid dehydrogenase type 3 and 17β-hydroxysteroid dehydrogenase type 5. Our group demonstrated that these enzymes are mainly expressed in mature adipocytes and that in women, trunk fat percentage is positively associated with adipose tissue mRNA abundance of both AKR1C2 and AKR1C3. In this context, our aim was to assess whether abdominal adipose tissue activity of AKR1C2 relates to excess visceral adiposity index (VAI) in women. Methods AKR1C2 activity was measured in visceral (VAT) and subcutaneous adipose tissue (SAT) of 31 women (age: 39 ± 7; BMI: 51 ± 6) undergoing bariatric surgery. Adipose tissue homogenate activity was measured by fluorimetry in a 12-hour kinetic experiment using the chemical compound cumberone, a competitive substrate in the inactivation of 5a-dihydrotestosterone to 3α-androstanediol by AKR1C2. Visceral adipose tissue excess was quantified using the previously published VAI equation and using the age-specific cut-offs for women, who were categorized into either high VAI or low VAI. Other markers of adipose tissue dysfunction included diacylglycerol acyltransferase 2 (DGAT2) and glutathione peroxidase 3 (GPX3) mRNA abundance, adipocyte size and pericellular fibrosis. Results Our technique allowed us to detect significant AKR1C2 activity in SAT (13.5±5.7 fluorescence units (FU)/min) and VAT (8.3±4.8 FU/min). Using the VAI equation, we identified 18 women with high VAI and 13 with low VAI. Women with high VAI were characterized by significantly higher VAT AKR1C2 activity when compared to women with low VAI (9.5 ± 4.4 vs. 6.4± 3.6 FU/min, p&amp;lt;0. 05). AKR1C2 mRNA abundance in either VAT or SAT was not significantly different as a function of VAI. Women with high VAI also had larger VAT adipocyte diameter and lower mRNA abundance of GPX3 in both adipose tissue depots compared to women with low VAI (p&amp;lt;0. 05 for all). No differences were seen in DGAT2 mRNA abundance, pericellular fibrosis or SAT adipocyte diameter as a function of VAI. Conclusion In women with severe obesity, excess visceral adiposity as indicated by a high VAI, is associated with higher AKR1C2 activity in VAT only. Similarly, adipose tissues of women with high VAI displayed features of adipose tissue dysfunction such as increased VAT adipocyte diameter and decreased GPX3expression in comparison to women with low VAI. Taken together our results confirm the link between adipose AKR1C2 and excess visceral adiposity. Presentation: No date and time listed

Establishment and Genetic Profiling of Platinum/Taxane Doublet-Resistant Cells Generated by Hybridizing Single Resistant Cells

Background: Current standard chemotherapy for gynecologic malignancies consists of platinum agent and taxane though, many patients experience the relapse of disease with drug resistance making the following therapy unsuccessful. It’s a compelling question whether the mechanisms of doubly resistance is a simple combination of single agent resistance or whether the core novel mechanism common to platinum and taxane resistance stands out as a result of combination therapy. The purpose of this study is to establish the doublet drug resistant cell line and to find its genetic characteristics. Methods: Platinum/taxane doublet resistant cell lines (F3 and F4) were generated by hybridizing two independent, platinum or taxane resistant subline (C13 or PX24) stemmed from grand parental ME180 cells. The resistant cells were selected through repeated exposure to cisplatin and paclitaxel. For the assessment of drug sensitivity, colony forming assay was used. For the gene expression analysis, genome-wide expression profiling was done using the Human Genome U133A Array. Protein-protein interaction network (PPI) scaffold networks were retrieved from the Search Tool for the Retrieval of Interacting Genes database and, for the enrichment of pathway analysis, WebGestalt was used. Results: Colony forming assay showed C13 was 5.8-fold cisplatin resistant while PX24 was 5.3-fold paclitaxel resistant compared with parental ME180 cells. F3 and F4 acquired resistance to cisplatin and paclitaxel by 8.3/4.9- and 3.7/3.3-fold (F3/4) respectively. Microarray analysis demonstrated, out of 22284 genes, 103 genes were &gt;4-fold up-regulated in F3/4 and 33 (32%) were identified as simultaneously upregulated genes (SUG) in C13, PX24 and F3/4. The Protein-protein interaction analysis of 33 SUG displayed a scaffold network pivoting aldo-keto reductase 1C1 (AKR1C1), aldo-keto reductase1C2 (AKR1C2) and aldo-keto reductase1C3 (AKR1C3). The enrichment pathway analysis demonstrated AKR1C gene family anchored to molecular function of oxidoreductase and aldo-keto reductase activity and biological process of daunorubicin and doxorubicin metabolism. Conclusions: We report here the establishment of doubly drug-resistant hybridoma to platinum and taxane. Analysis of SUG indicated the AKR1C gene family plays a key role for doubly resistant mechanism that would be possible targets for therapeutic strategies.

Bruton's Tyrosine Kinase Inhibitor Zanubrutinib Effectively Modulates Cancer Resistance by Inhibiting Anthracycline Metabolism and Efflux.

Zanubrutinib (ZAN) is a Bruton’s tyrosine kinase inhibitor recently approved for the treatment of some non-Hodgkin lymphomas. In clinical trials, ZAN is often combined with standard anthracycline (ANT) chemotherapy. Although ANTs are generally effective, drug resistance is a crucial obstacle that leads to treatment discontinuation. This study showed that ZAN counteracts ANT resistance by targeting aldo-keto reductase 1C3 (AKR1C3) and ATP-binding cassette (ABC) transporters. AKR1C3 catalyses the transformation of ANTs to less potent hydroxy-metabolites, whereas transporters decrease the ANT-effective concentrations by pumping them out of the cancer cells. In our experiments, ZAN inhibited the AKR1C3-mediated inactivation of daunorubicin (DAUN) at both the recombinant and cellular levels. In the drug combination experiments, ZAN synergistically sensitised AKR1C3-expressing HCT116 and A549 cells to DAUN treatment. Gene induction studies further confirmed that ZAN did not increase the intracellular level of AKR1C3 mRNA; thus, the drug combination effect is not abolished by enzyme induction. Finally, in accumulation assays, ZAN was found to interfere with the DAUN efflux mediated by the ABCB1, ABCG2, and ABCC1 transporters, which might further contribute to the reversal of ANT resistance. In summary, our data provide the rationale for ZAN inclusion in ANT-based therapy and suggest its potential for the treatment of tumours expressing AKR1C3 and/or the above-mentioned ABC transporters.