Paracrine interactions between adipose fibroblasts and malignant epithelial cells are essential for structural and hormonal support of breast tumors. Factors derived from malignant epithelial cells ...inhibit adipogenic differentiation of fibroblasts and upregulate expression of aromatase, which stimulates estrogen synthesis and creates a localized, growth-stimulatory environment. Here, we characterized the gene expression profile of breast adipose fibroblasts in an
in vitro model of malignancy to identify other paracrine interactions that support tumor growth. Primary breast adipose fibroblasts from cancer-free women were treated with conditioned media from malignant breast epithelial cells or normal breast epithelial cells, and differences in gene expression were identified by microarray. A total of 79 differentially regulated genes encoding cytokines, enzymes, angiogenic factors, cytoskeletal proteins, extra-cellular matrix remodeling proteins, signal transduction proteins and cell surface receptors were identified, and 6 of these were verified by real-time PCR. Among these, the expression of aldo-keto reductase family 1, member C3 (AKR1C3) was upregulated. AKR1C3 has multiple enzymatic properties, including conversion of estrone to estradiol and androstenedione to testosterone. Immunoreactive AKR1C3 was detected in epithelial and stromal components of benign lesions and ductal carcinomas
in situ, and in 59.8% of epithelial and 69.6% of stromal cells in invasive breast carcinomas. AKR1C3 expression was significantly higher in myoepithelial cells surrounding the neoplastic epithelium of ductal carcinoma
in situ compared with those surrounding benign epithelial lesions. Importantly, AKR1C3 and aromatase mRNA levels correlated positively in 61 malignant breast tumors (
R
=
0.3967,
p
=
0.00156). Malignant epithelial cell-conditioned medium significantly increased formation of testosterone and estradiol from androstenedione in breast adipose fibroblasts. In conclusion, malignant epithelial cell-derived factors significantly upregulate the enzymes AKR1C3 and aromatase that catalyze a series of complementary reactions to convert the circulating precursor androstenedione to biologically active estradiol
in vitro in the stromal fibroblasts, and
in vivo, in stromal component of breast tumors.
Structure of AKR1C3 with 3-phenoxybenzoic acid bound Jackson, Victoria J.; Yosaatmadja, Yuliana; Flanagan, Jack U. ...
Acta crystallographica. Section F, Structural biology and crystallization communications,
April 2012, Volume:
68, Issue:
4
Journal Article
Peer reviewed
Open access
Aldo–keto reductase 1C3 (AKR1C3) is a human enzyme that catalyzes the NADPH‐dependent reduction of steroids and prostaglandins. AKR1C3 overexpression is associated with the proliferation of ...hormone‐dependent cancers, most notably breast and prostate cancers. Nonsteroidal anti‐inflammatory drugs (NSAIDs) and their analogues are well characterized inhibitors of AKR1C3. Here, the X‐ray crystal structure of 3‐phenoxybenzoic acid in complex with AKR1C3 is presented. This structure provides useful information for the future development of new anticancer agents by structure‐guided drug design.
3α-Hydroxysteroid dehydrogenase (3α-HSD) isoforms (AKR1C1–AKR1C4) are aldo-keto reductases that metabolize steroids and other substances in many tissues including the CNS. Here we demonstrated that ...in glaucomatous human optic nerve heads, increased expression of 3α-HSD was localized to reactive astrocytes in the lamina cribrosa. Similar, optic nerve head astrocytes exhibited increased expression of 3α-HSD in response to elevated intraocular pressure in a monkey model of experimental glaucoma, but not in monkeys with unilateral optic nerve transection. In vitro, glaucomatous optic nerve head astrocytes expressed higher levels of AKR1C1, AKR1C2, and AKR1C3 mRNA, than normal astrocytes, with significant differential increase of AKR1C2 expression, and exhibited higher enzymatic activity forming 3α-androstanediol a well-recognized neurosteroid. Normal astrocytes exposed to elevated hydrostatic pressure selectively increased AKR1C2 expression. Our findings of increased expression of 3α-HSDs in glaucomatous optic nerve head astrocytes offer new insights into possible roles for neurosteroids in the pathophysiology of glaucoma.
The chemopreventive activity of sulforaphane (SFN) occurs through its inhibition of carcinogen-activating enzymes and its induction of detoxification enzymes. However, the exact mechanisms by which ...SFN exerts its anti-carcinogenic effects are not fully understood. Therefore, the mechanisms underlying the cytoprotective effects of SFN were examined in MCF-7 breast cancer cells. Exposure of cells to SFN (10 μM) induced a transcriptional change in the AKR1C3 gene, which is one of aldo-keto reductases (AKRs) family that is associated with detoxification and antioxidant response. Further analysis revealed that SFN elicited a dose- and time-dependent increase in the expression of both the NRF2 and AKR1C3 proteins. Moreover, this up-regulation of AKR1C3 was inhibited by pretreatment with antioxidant, N-acetyl-L-cysteine (NAC), which suggests that the up-regulation of AKR1C3 expression induced by SFN involves reactive oxygen species (ROS) signaling. Furthermore, pretreatment of cells with LY294002, a pharmacologic inhibitor of phosphatidylinositol 3-kinase (PI3K), suppressed the SFN-augmented Nrf2 activation and AKR1C3 expression; however, inhibition of PKC or MEK1/2 signaling with Go6976 or PD98059, respectively, did not alter SFN-induced AKR1C3 expression. Collectively, these data suggest that SFN call modulate the expression of the AKR1C3 in MCF-7 cells by activation of PI3K via the generation of ROS.
Chemotherapy is typically used to treat choriocarcinoma, but a small proportion of tumors develop resistance to chemotherapy. Similarly, methotrexate (MTX) is a first-line chemotherapy used to treat ...choriocarcinoma; although ~30% of patients are drug-resistant for MTX mono-therapy. Thus, we sought to elucidate the mechanism of chemotherapeutic-resistance of MTX.
RNA interference technology, colony formation, and MTT assays were used to investigate the role of aldo-keto reductase family 1, member C3 (AKR1C3) in MTX resistance in choriocarcinoma cells.
AKR1C3 expression was higher in JeG-3R cells compared to JeG-3 cells and targeted inhibition of AKR1C3 expression with shRNA suppresses growth of choriocarcinoma cells as measured by colony formation and MTT assays. Overexpression of AKR1C3 increased chemotherapeutic resistance in JeG-3 cells. Furthermore, AKR1C3 silencing increases sensitivity to MTX in JeG-3R choriocarcinoma cells. Increasing MTX sensitivity spears to be related to DNA damage induction by increased reactive oxygen species (ROS), apoptosis, and cell cycle arrest.
Data show that AKR1C3 is critical to the development of methotrexate resistance in choriocarcinoma and suggest that AKR1C3 may potentially serve as a therapeutic marker for this disease.
Human 3 alpha -hydroxysteroid dehydrogenase exists in four isoforms, which belong to the aldo-keto reductase (AKR) superfamily and are named AKR1C1-AKR1C4. The properties of AKR1C3 have not been ...fully characterized compared to the other isoforms. In addition, a cDNA that shows more than 99% homology with AKR1C3 cDNA has been cloned from human myeloblasts. We have here expressed and purified a recombinant enzyme (designated as DBDH) from this cDNA. DBDH oxidized xenobiotic alicyclic alcohols and 3 alpha - or 17 beta -hydroxy-5 beta -androstanes, and catalyzed the reversible conversion between prostaglandin D sub(2) and 9 alpha , 11 beta -prostaglandin F sub(2) more efficiently than that of 3 alpha - or 17 beta -hydroxysteroids: the respective K sub(m) values were 0.6 and 6.8 mu M, and k sub(cat)/K sub(m) values were about 1,000 min super(-1) times mM super(-1). Anti-inflammatory drugs highly inhibited the enzyme. The recombinant AKR1C3 prepared by site-directed mutagenesis of DBDH also showed the same properties as the wild-type DBDH. Analyses of expression of mRNAs for DBDH and AKR1C3 by reverse transcription-PCR indicated that only one mRNA species for DBDH is expressed in 33 human specimens of liver, kidney, lung, brain, heart, spleen, adrenal gland, small intestine, placenta, prostate, and testis. These results suggest that AKR1C3 acts as prostaglandin D sub(2) 11-ketoreductase, and that its principal gene in the human has a coding region represented by DBDH cDNA.
Rat liver 3α-hydroxysteroid/dihydrodiol dehydrogenase (3α-HSD/DD) is a member of the aldo-keto reductase (AKR) superfamily. It is involved in the inactivation of steroid hormones and the metabolic ...activation of polycyclic aromatic hydrocarbons (PAH) by converting trans-dihydrodiols into reactive and redox-active o-quinones. The structure of the 5′-flanking region of the gene and factors involved in the constitutive and regulated expression of this gene have been reported H.-K. Lin, T.M. Penning, Cloning, sequencing, and functional analysis of the 5′-flanking region of the rat 3α-hydroxysteroid/dihydrodiol dehydrogenase gene, Cancer Res. 55 (1995) 4105–4113. We now describe the complete genomic structure of the rat type 1 3α-HSD/DD gene. Charon 4A and P1 genomic clones contained at least three rat genes (type 1, type 2 and type 3 3α-HSD/DD) each of which encoded for the same open reading frame (ORF) but differed in their exon–intron organization. 5′-RACE confirmed that the type 1 3α-HSD/DD gene encodes for the dominant transcript in rat liver and it was the regulation of this gene that was previously studied. The rat type 1 3α-HSD/DD gene is 30 kb in length and consists of nine exons and eight introns. Exon 9 encodes +931 to 966 bp of the ORF and the 1292 bp 3′-UTR implicated in mRNA stability. This genomic structure is nearly identical to the homologous human genes, type 1 3α-HSD (chlordecone reductase/DD4, AKR1C4), type 2 3α-HSD (AKR1C3) and type 3 3α-HSD (bile-acid binding protein, AKR1C2) genes. Three different cDNA's containing identical ORFs for 3α-HSD have been reported suggesting that all three genes may be expressed in rat liver. Using 5′ primers corresponding to the 5′-UTR's of the three different cDNA's only one PCR fragment was obtained and corresponded to the type 1 3α-HSD/DD gene. These data suggested that the type 2 and type 3 3α-HSD/DD genes are not abundantly expressed in rat liver. It is unknown whether the type 2 and type 3 3α-HSD/DD genes represent pseudo-genes or whether they represent genes that are differentially expressed in other rat tissues.
The aldo-keto reductase AKR1C3, has been shown to regulate myelopoiesis via its ability to metabolise prostaglandin D2 (PGD2). Other studies have demonstrated the oxidative activation of polycyclic ...aromatic hydrocarbon (PAH) procarcinogens by AKR1C3 in cell-free systems. This is the first study that addresses whether AKR1C3 mediates carcinogen activation within intact living cells following manipulation of AKR1C3 by molecular intervention. Quantitative RT-PCR identified AKR1C3 as the predominant AKR1C isoform expressed in acute myeloid leukemia (AML). Exposure of K562 and KG1a myeloid cell lines to the known AKR1C3 substrate 7,12-dimethylbenz(a)anthracene-3,4-dihydrodiol (7,12-DMBA-3,4-diol) resulted in both single strand DNA breaks and oxidative DNA damage as measured using conventional and FPG-modified comet assays respectively. PGD2-keto reductase activity was shown to be correlated with relative AKR1C3 expression and together with quantitative real time PCR was used to validate the RNAi-knockdown of AKR1C3 in K562 cells. Knockdown of AKR1C3 did not alter single strand DNA breaks following 7,12-DMBA-3,4-diol exposure but significantly decreased oxidative DNA damage. A similar interrelationship between AKR1C3 activity and 7,12-DMBA-3,4-diol mediated oxidative DNA damage but not single strand breaks was observed in KG1a cells. Finally, AKR1C3 knockdown also resulted in spontaneous erythroid differentiation of K562 cells. Since K562 cells are a model of AML blast crisis of chronic myeloid leukemia (CML) the data presented here identify AKR1C3 as a novel mediator of carcinogen-induced initiation of leukemia, as a novel regulator of erythroid differentiation and paradoxically as a potential new target in the treatment of CML.
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