Correlation of aldo-ketoreductase (AKR) 1C3 genetic variant with doxorubicin pharmacodynamics in Asian breast cancer patients

Aims

Aldo-ketoreductases have been implicated in the metabolism of doxorubicin. We sought to assess the influence of AKR1C3 genetic variants on doxorubicin metabolism.

Methods

We sequenced AKR1C3 exon 5 and genotyped seven functional single nucleotide polymorphisms in CBR3, ABCB1 and SLC22A16 involved in doxorubicin pharmacology in 151 Asian breast cancer patients treated with doxorubicin-containing chemotherapy, and correlated these genotypes with doxorubicin pharmacokinetics and pharmacodynamics.

Results

Two previously reported AKR1C3 intronic variants, IVS4–212 C>G and IVS4+218 G>A, were detected. The AKR1C3 IVS4–212 GG genotype was associated with significantly lower cycle 1 day 15 leucocyte (mean leucocytes 2.49 ± 1.57 × 10⁹ vs. 3.85 ± 3.42 × 10⁹ l⁻¹, P = 0.007) and neutrophil counts (mean neutrophils 0.70 ± 1.01 × 10⁹ vs. 1.56 ± 2.80 × 10⁹ l⁻¹, P = 0.008) and significant improvement of progression-free survival [PFS, mean PFS 49.0 (95% confidence interval 42.2–55.8) vs. 31.0 (95% confidence interval 20.7–41.2) months, P = 0.017] and overall survival [OS; mean OS 64.4 (95% confidence interval 58.3–70.5) vs. 46.3 (95% confidence interval 35.1–57.5) months, P = 0.006] compared with those carrying at least one C allele. There was no significant association between AKR1C3 IVS4–212 C>G and doxorubicin pharmacokinetics. Of the other seven single nucleotide polymorphisms genotyped, CBR3 G11A correlated with doxorubicinol area under the concentration–time curve and OS, ABCB1 G2677T/A correlated with doxorubicin clearance and platelet toxicity, while ABCB1 IVS26+59 T>G correlated with OS. The AKR1C3 IVS4–212 C<G genotype remained significantly correlated with both PFS and OS on multivariate analysis with clinical prognosticators.

Conclusions

The AKR1C3 IVS4–212 GG genotype was associated with greater haematological toxicity and longer progression-free survival and overall survival after doxorubicin-based therapy, suggesting potential interaction of this variant with doxorubicin metabolism.     

AKR1B10 induces cell resistance to daunorubicin and idarubicin by reducing C13 ketonic group

Daunorubicin, idarubicin, doxorubicin and epirubicin are anthracyclines widely used for the treatment of lymphoma, leukemia, and breast, lung, and liver cancers, but tumor resistance limits their clinical success. Aldo-keto reductase family 1 B10 (AKR1B10) is an NADPH-dependent enzyme overexpressed in liver and lung carcinomas. This study was aimed to determine the role of AKR1B10 in tumor resistance to anthracyclines. AKR1B10 activity toward anthracyclines was measured using recombinant protein. Cell resistance to anthracycline was determined by ectopic expression of AKR1B10 or inhibition by epalrestat. Results showed that AKR1B10 reduces C13-ketonic group on side chain of daunorubicin and idarubicin to hydroxyl forms. In vitro, AKR1B10 converted daunorubicin to daunorubicinol at V_max of 837.42 ± 81.39 nmol/mg/min, K_m of 9.317 ± 2.25 mM and k_cat/K_m of 3.24. AKR1B10 showed better catalytic efficiency toward idarubicin with V_max at 460.23 ± 28.12 nmol/mg/min, K_m at 0.461 ± 0.09 mM and k_cat/K_m at 35.94. AKR1B10 was less active toward doxorubicin and epirubicin with a C14-hydroxyl group. In living cells, AKR1B10 efficiently catalyzed reduction of daunorubicin (50 nM) and idarubicin (30 nM) to corresponding alcohols. Within 24 h, approximately 20 ± 2.7% of daunorubicin (1 μM) or 23 ± 2.3% of idarubicin (1 μM) was converted to daunorubicinol or idarubicinol in AKR1B10 expression cells compared to 7 ± 0.9% and 5 ± 1.5% in vector control. AKR1B10 expression led to cell resistance to daunorubicin and idarubicin, but inhibitor epalrestat showed a synergistic role with these agents. Together our data suggest that AKR1B10 participates in cellular metabolism of daunorubicin and idarubicin, resulting in drug resistance. These data are informative for the clinical use of idarubicin and daunorubicin.     

Investigations on the cytochrome P450 (CYP) isoenzymes involved in the metabolism of the designer drugs N-(1-phenyl cyclohexyl)-2-ethoxyethanamine and N-(1-phenylcyclohexyl)-2-methoxyethanamine

Investigations using insect cell microsomes with cDNA-expressed human cytochrome P450 (CYP)s and human liver microsomes (HLM) are reported on the CYP isoenzymes involved in the metabolism of the designer drugs N-(1-phenylcyclohexyl)-2-ethoxyethanamine (PCEEA) to O-deethyl PCEEA and N-(1-phenylcyclohexyl)-2-methoxyethanamine (PCMEA) to O-demethyl PCMEA. Gas chromatography-mass spectrometry or liquid chromatography-mass spectrometry was used for the analysis of the incubation samples. PCEEA O-deethylation was catalyzed by CYP2B6, CYP2C9, CYP2C19, and CYP3A4, while PCMEA O-demethylation was catalyzed only by CYP2B6 and CYP2C19. Considering the relative activity factor approach, these enzymes accounted for 53%, 25%, 4%, and 18% of net clearance for PCEEA and 91% and 9% of net clearance for PCMEA, respectively. The chemical CYP2B6 inhibitor 4-(4-chlorobenzyl)pyridine (CBP) reduced the metabolite formation in pooled HLM by 63% at 1 μM PCEEA. At 10 μM PCEEA, CBP reduced metabolite formation by 61%, while inhibition of CYP3A4 by ketoconazole and inhibition of CYP2C9 by sulfaphenazole showed no inhibitory effect. At 1 μM PCMEA, CBP reduced metabolite formation in pooled HLM by 70% and at 10 μM PCMEA by 78%, respectively. In conclusion, the main metabolic step of both studied drugs was catalyzed by different CYPs.     

Growth Inhibitory Effect of (E)-2,4-bis(p-hydroxyphenyl)-2-Butenal Diacetate through Induction of Apoptotic Cell Death by Increasing DR3 Expression in Human Lung Cancer Cells

The Maillard Reaction Products (MRPs) are chemical compounds which have been known to be effective in chemoprevention. Death receptors (DR) play a central role in directing apoptosis in several cancer cells. In our previous study, we demonstrated that (E)-2,4-bis(p-hydroxyphenyl)-2-butenal, a MRP product, inhibited human colon cancer cell growth by inducing apoptosis via nuclear factor-κB (NF-κB) inactivation and G₂/M phase cell cycle arrest. In this study, (E)-2,4-bis(p-hydroxyphenyl)-2-butenal diacetate, a new (E)-2,4-bis(p-hydroxyphenyl)-2-butenal derivative, was synthesized to improve their solubility and stability in water and then evaluated against NCI-H460 and A549 human lung cancer cells. (E)-2,4-bis(p-hydroxyphenyl)-2-butenal diacetate reduced the viability in both cell lines in a time and dose-dependent manner. We also found that (E)-2,4-bis(p-hydroxyphenyl)-2-butenal diacetate increased apoptotic cell death through the upregulation of the expression of death receptor (DR)-3 and DR6 in both lung cancer cell lines. In addition to this, the transfection of DR3 siRNA diminished the growth inhibitory and apoptosis inducing effect of (E)-2,4-bis(p-hydroxyphenyl)-2-butenal diacetate on lung cancer cells, however these effects of (E)-2,4-bis(p-hydroxyphenyl)-2-butenal diacetate was not changed by DR6 siRNA. These results indicated that (E)-2,4-bis(p-hydroxyphenyl)-2-butenal diacetate inhibits human lung cancer cell growth via increasing apoptotic cell death by upregulation of the expression of DR3.     

The combined impact of CYP2C19 and CYP2B6 pharmacogenetics on cyclophosphamide bioactivation

AIMS

The role of CYP pharmacogenetics in the bioactivation of cyclophosphamide is still controversial. Recent clinical studies have suggested a role for either CYP2C19 or CYP2B6. The aim of this study was to clarify the role of these pharmacogenes.

METHODS

We used a combined in vitro–in vivo approach to determine the role of these pharmacogenes in the bioactivation of the prodrug to 4-hydroxy cyclophosphamide (4-OHCP). Cyclophosphamide metabolism was determined in a human liver biobank (n = 14) and in patients receiving the drug for treatment of lupus nephritis (n = 16)

RESULTS

In livers of known CYP2C19 and CYP2B6 genotype and protein expression we observed that there was a combined role for both CYP2C19 and CYP2B6 in the bioactivation of cyclophosphamide in vitro. The presence of at least one loss of function (LoF) allele at either CYP2C19 or CYP2B6 resulted in a significant decrease in both V_max (P = 0.028) and CL_int (P = 0.0017) compared with livers with no LoF alleles. This dual genotype relationship was also observed in a preliminary clinical study, with patients who had ≥1 LoF allele at either CYP2C19 or CYP2B6 also displaying significantly (P = 0.0316) lower bioactivation of cyclophosphamide. The mean 4-OHCP : CP bioactivation ratio was 0.0014 (95% CI 0.0007, 0.002) compared with 0.0071 (95% CI 0.0001, 0.014) in patients with no LoF alleles at either of these genes.

CONCLUSIONS

The presence of ≥1 LoF allele(s) at either CYP2B6 or CYP2C19 appeared to result in decreased bioactivation of cyclophosphamide both in vitro and in patients. Further clinical studies to confirm this relationship are warranted.     

An indomethacin analogue, N-(4-chlorobenzoyl)-melatonin, is a selective inhibitor of aldo-keto reductase 1C3 (type 2 3alpha-HSD, type 5 17beta-HSD, and prostaglandin F synthase), a potential target for the treatment of hormone dependent and hormone independent malignancies

Aldo-keto reductase (AKR) 1C3 (type 2 3alpha-HSD, type 5 17beta-HSD, and prostaglandin F synthase) regulates ligand access to steroid hormone and prostaglandin receptors and may stimulate proliferation of prostate and breast cancer cells. NSAIDs are known inhibitors of AKR1C enzymes. An NSAID analogue that inhibits AKR1C3 but is inactive against the cyclooxygenases and the other AKR1C family members would provide an important tool to examine the role of AKR1C3 in proliferative signaling. We tested NSAIDs and NSAID analogues for inhibition of the reduction of 9,10-phenanthrenequinone (PQ) catalyzed by AKR1C3 and the closely related isoforms AKR1C1 and AKR1C2. Two of the compounds initially screened, indomethacin and its methyl ester, were specific for AKR1C3 versus the other AKR1C isoforms. Based on these results and the crystal structure of AKR1C3, we predicted that N-(4-chlorobenzoyl)-melatonin (CBM), an indomethacin analogue that does not inhibit the cyclooxygenases, would selectively inhibit AKR1C3. CBM inhibited the reduction of PQ by AKR1C3, but did not significantly inhibit AKR1C1 or AKR1C2. Indomethacin and CBM also inhibited the AKR1C3-catalyzed reduction of Delta(4)-androstene-3,17-dione but did not significantly inhibit the reduction of steroid hormones catalyzed by AKR1C1 or AKR1C2. The pattern of inhibition of AKR1C3 by indomethacin and CBM was uncompetitive versus PQ, but competitive versus Delta(4)-androstene-3,17-dione, indicating that two different inhibitory complexes form during the ordered bi bi reactions. The identification of CBM as a specific inhibitor of AKR1C3 will aid the investigation of its roles in steroid hormone and prostaglandin signaling and the resultant effects on cancer development.     

A new nomenclature for the aldo-keto reductase superfamily

The aldo-keto reductases (AKRs) represent a growing oxidoreductase superfamily. Forty proteins have been identified and characterized as AKRs, and an additional fourteen genes may encode proteins related to the superfamily. Found in eukaryotes and prokaryotes, the AKRs metabolize a wide range of substrates, including aliphatic aldehydes, monosaccharides, steroids, prostaglandins, and xenobiotics. This broad substrate specificity has caused problems in naming these proteins. Enzymes capable of these reactions have been referred to as aldehyde reductase (ALR1), aldose reductase (ALR2), and carbonyl reductase (ALR3); however, ALR3 is not a member of the AKR superfamily. Also, some AKRs have multiple names based upon substrate specificity. For example, human 3α-hydroxysteroid dehydrogenase (3α-HSD) type I is also known as dihydrodiol dehydrogenase 4 and chlordecone reductase. To address these issues, we propose a new nomenclature system for the AKR superfamily based on amino acid sequence identities. Cluster analysis of the AKRs shows seven distinct families at the 40% amino acid identity level. The largest family (AKR1) contains the aldose reductases, aldehyde reductases, and HSDs. Other families include the prokaryotic AKRs, the plant chalcone reductases, the Shaker channels, and the ethoxyquin-inducible aflatoxin B₁ aldehyde reductase. At the level of 60% amino acid identity, subfamilies are discernible. For example, the AKR1 family includes five subfamilies: (A) aldehyde reductases (mammalian); (B) aldose reductases; (C) HSDs; (D) Δ⁴-3-ketosteroid-5β-reductases; and (E) aldehyde reductases (plant). This cluster analysis forms the basis for our nomenclature system. Recommendations for naming an aldo-keto reductase include the root symbol “AKR,” an Arabic number designating the family, a letter indicating the subfamily when multiple subfamilies exist, and an Arabic numeral representing the unique protein sequence. For example, human aldehyde reductase would be assigned as AKR1A1. Our nomenclature is both systematic and expandable, thereby allowing assignment of consistent designations for newly identified members of the superfamily.     

五味子乙素对HK-2细胞缺氧损伤的保护作用

卢爱龙 1, 谭小月 2, 张勉之 3△, 吴银娜摘要： 目的 探讨五味子乙素 （Sch B） 对氯化钴 （CoCl2） 诱导的人类近端肾小管上皮 （HK-2） 细胞缺氧损伤的保护作用及其可能机制。方法 取离体培养 HK-2 细胞, 随机分为 4 组。对照 （C） 组： 细胞未经任何处理。CoCl2组 （化学乏氧组）： 加入 600 μmol/L 的 CoCl2 处理 24 h。Sch B 预保护（CoCl2+ Sch B）组： 分别加入终浓度为 1 μmol/L 和 10 μmol/L Sch B 预处理 2 h 后, 其余操作同 CoCl2 组。Sch B 组： 分别加入终浓度 1 μmol/L 和 10 μmol/L Sch B 处理 2 h。CCK-8 试剂盒检测各组细胞活性; AnnexinV-FITC/PI 双标记流式细胞仪检测各组细胞凋亡率; Western Blot 检测各组缺氧诱导因子-1α（HIF-1α）蛋白表达; RT-PCR 检测各组 HIF-1α和诱导型一氧化氮合酶（iNOS） mRNA 表达。结果 与对照组相比, CoCl2组细胞活性明显降低, 细胞凋亡率、 HIF-1α蛋白表达量和 iNOS mRNA 表达量显著增加, HIF-1α mRNA 表达量差异无统计学意义; Sch B 预保护组较 CoCl2组细胞活性显著增加, 细胞凋亡率、 HIF-1α蛋白表达量、 HIF-1α及 iNOS mRNA 表达量均显著减少; Sch B 组与对照组细胞活性、 细胞凋亡率差异无统计学意义, Sch B 组几乎不表达 HIF-1α蛋白。结论 Sch B 可能通过抑制 HIF-1α蛋白和 iNOS mRNA 的表达减少 HK-2 细胞的凋亡, 从而对 HK-2 细胞缺氧损伤起保护作用。     

CYP1A1 and CYP1A2 expression: Comparing ‘humanized’ mouse lines and wild-type mice; comparing human and mouse hepatoma-derived cell lines

Human and rodent cytochrome P450 (CYP) enzymes sometimes exhibit striking species-specific differences in substrate preference and rate of metabolism. Human risk assessment of CYP substrates might therefore best be evaluated in the intact mouse by replacing mouse Cyp genes with human CYP orthologs; however, how “human-like” can human gene expression be expected in mouse tissues? Previously a bacterial-artificial-chromosome-transgenic mouse, carrying the human CYP1A1_CYP1A2 locus and lacking the mouse Cyp1a1 and Cyp1a2 orthologs, was shown to express robustly human dioxin-inducible CYP1A1 and basal versus inducible CYP1A2 (mRNAs, proteins, enzyme activities) in each of nine mouse tissues examined. Chimeric mice carrying humanized liver have also been generated, by transplanting human hepatocytes into a urokinase-type plasminogen activator(+/+)_severe-combined-immunodeficiency (uPA/SCID) line with most of its mouse hepatocytes ablated. Herein we compare basal and dioxin-induced CYP1A mRNA copy numbers, protein levels, and four enzymes (benzo[a]pyrene hydroxylase, ethoxyresorufin O-deethylase, acetanilide 4-hydroxylase, methoxyresorufin O-demethylase) in liver of these two humanized mouse lines versus wild-type mice; we also compare these same parameters in mouse Hepa-1c1c7 and human HepG2 hepatoma-derived established cell lines. Most strikingly, mouse liver CYP1A1-specific enzyme activities are between 38- and 170-fold higher than human CYP1A1-specific enzyme activities (per unit of mRNA), whereas mouse versus human CYP1A2 enzyme activities (per unit of mRNA) are within 2.5-fold of one another. Moreover, both the mouse and human hepatoma cell lines exhibit striking differences in CYP1A mRNA levels and enzyme activities. These findings are relevant to risk assessment involving human CYP1A1 and CYP1A2 substrates, when administered to mice as environmental toxicants or drugs.     

The effect of acetaminophen on the expression of BCRP in trophoblast cells impairs the placental barrier to bile acids during maternal cholestasis

Acetaminophen is used as first-choice drug for pain relief during pregnancy. Here we have investigated the effect of acetaminophen at subtoxic doses on the expression of ABC export pumps in trophoblast cells and its functional repercussion on the placental barrier during maternal cholestasis. The incubation of human choriocarcinoma cells (JAr, JEG-3 and BeWo) with acetaminophen for 48 h resulted in no significant changes in the expression and/or activity of MDR1 and MRPs. In contrast, in JEG-3 cells, BCRP mRNA, protein, and transport activity were reduced. In rat placenta, collected at term, acetaminophen administration for the last three days of pregnancy resulted in enhanced mRNA, but not protein, levels of Mrp1 and Bcrp. In fact, a decrease in Bcrp protein was found. Using in situ perfused rat placenta, a reduction in the Bcrp-dependent fetal-to-maternal bile acid transport after treating the dams with acetaminophen was found. Complete biliary obstruction in pregnant rats induced a significant bile acid accumulation in fetal serum and tissues, which was further enhanced when the mothers were treated with acetaminophen. This drug induced increased ROS production in JEG-3 cells and decreased the total glutathione content in rat placenta. Moreover, the NRF2 pathway was activated in JEG-3 cells as shown by an increase in nuclear NRF2 levels and an up-regulation of NRF2 target genes, NQO1 and HMOX-1, which was not observed in rat placenta. In conclusion, acetaminophen induces in placenta oxidative stress and a down-regulation of BCRP/Bcrp, which may impair the placental barrier to bile acids during maternal cholestasis.     

Exposure to 9,10-phenanthrenequinone accelerates malignant progression of lung cancer cells through up-regulation of aldo-keto reductase 1B10

Inhalation of 9,10-phenanthrenequinone (9,10-PQ), a major quinone in diesel exhaust, exerts fatal damage against a variety of cells involved in respiratory function. Here, we show that treatment with high concentrations of 9,10-PQ evokes apoptosis of lung cancer A549 cells through production of reactive oxygen species (ROS). In contrast, 9,10-PQ at its concentrations of 2 and 5 μM elevated the potentials for proliferation, invasion, metastasis and tumorigenesis, all of which were almost completely inhibited by addition of an antioxidant N-acetyl-l-cysteine, inferring a crucial role of ROS in the overgrowth and malignant progression of lung cancer cells. Comparison of mRNA expression levels of six aldo-keto reductases (AKRs) in the 9,10-PQ-treated cells advocated up-regulation of AKR1B10 as a major cause contributing to the lung cancer malignancy. In support of this, the elevation of invasive, metastatic and tumorigenic activities in the 9,10-PQ-treated cells was significantly abolished by the addition of a selective AKR1B10 inhibitor oleanolic acid. Intriguingly, zymographic and real-time PCR analyses revealed remarkable increases in secretion and expression, respectively, of matrix metalloproteinase 2 during the 9,10-PQ treatment, and suggested that the AKR1B10 up-regulation and resultant activation of mitogen-activated protein kinase cascade are predominant mechanisms underlying the metalloproteinase induction. In addition, HPLC analysis and cytochrome c reduction assay in in vitro 9,10-PQ reduction by AKR1B10 demonstrated that the enzyme catalyzes redox-cycling of this quinone, by which ROS are produced. Collectively, these results suggest that AKR1B10 is a key regulator involved in overgrowth and malignant progression of the lung cancer cells through ROS production due to 9,10-PQ redox-cycling.