This study was conducted to determine whether the ratio of estrogen-DNA adducts to their respective metabolites and conjugates in serum differed between women with early-onset breast cancer and those with average or high risk of developing breast cancer.
Serum samples from women at average risk (n = 63) or high risk (n = 80) for breast cancer (using Gail model) and women newly diagnosed with early breast cancer (n = 79) were analyzed using UPLC-MS/MS. Adduct ratios were statistically compared among the three groups, and the Area Under the Receiver Operating Characteristic Curve (AUC) was used to identify a diagnostic cut-off point.
The median adduct ratio in the average-risk group was significantly lower than that of both the high-risk group and the breast cancer group (p values <0.0001), and provided good discrimination between those at average versus high risk of breast cancer (AUC = 0.84, 95% CI 0.77–0.90). Sensitivity and specificity were maximized at an adduct ratio of 77. For women in the same age and BMI group, the odds of being at high risk for breast cancer was 8.03 (95% CI 3.46–18.7) times higher for those with a ratio of at least 77 compared to those with a ratio less than 77.
The likelihood of being at high risk for breast cancer was significantly increased for those with a high adduct ratio relative to those with a low adduct ratio. These findings suggest that estrogen-DNA adducts deserve further study as potential biomarkers for risk of developing breast cancer.
breast cancer; depurinating estrogen-DNA adducts; biomarker; breast cancer prevention
The nuclear vitamin D receptor (VDR) modulates gene transcription in 1,25-dihydroxyvitamin D3 (1,25D) target tissues such as kidney, intestine, and bone. VDR is also expressed in heart, and 1,25D deficiency may play a role in the acceleration of cardiovascular disease. Employing a yeast two-hybrid system and a human heart library, using both a 1,25D-independent and 1,25D-dependent screen, we discovered six candidate VDR interacting proteins (VIPs). These novel VIPs include CXXC5, FASTK, NR4A1, TPM2, MYL3 and XIRP1. Mammalian two-hybrid assays as well as GST pull-downs were used to confirm VIP-VDR interaction, and the combination of these two assays reveals that CXXC5, XIRP1, FASTK and NR4A1 interactions with VDR may be modulated by 1,25D. The functional effects of these VIPs on 1,25D-mediated gene expression were explored in transcriptional assays employing three separate and distinct 1,25D-responsive element (VDRE)-driven luciferase reporter genes in transfected Caco-2 and HEK-293 cells, and in a C2C12 myoblast line. FASTK and TPM2 activated expression in all cell line and promoter contexts, while CXXC5 and XIRP1 exhibited differing effects depending on the cell line and promoter employed, suggesting promoter and cell-specific effects of these unique VIPs on VDR signaling. Further evaluation of the interaction between CXXC5 and VDR revealed that CXXC5 acts in a dose-dependent manner to stimulate VDR-mediated transcription on select VDREs. Identification of novel heart VIPs and their influence on VDR activity may increase our understanding of how vitamin D impacts cardiac physiology and may facilitate development of VDR/VIP drug analogs to combat heart disease.
vitamin D; vitamin D receptor; vitamin D responsive elements; transcription; co-modulators; CXXC5
Estrogen plays important roles in hormone receptor-positive breast cancer. Endocrine therapies, such as the antiestrogen tamoxifen, antagonize the binding of estrogen to estrogen receptor (ER), whereas aromatase inhibitors (AIs) directly inhibit the production of estrogen. Understanding the mechanisms of endocrine resistance and the ways in which we may better treat these types of resistance has been aided by the development of cellular models for resistant breast cancers. In this review, we will discuss what is known thus far regarding both de novo and acquired resistance to tamoxifen or AIs. Our laboratory has generated a collection of AI- and tamoxifen-resistant cell lines in order to comprehensively study the individual types of resistance mechanisms. Through the use of microarray analysis, we have determined that our cell lines resistant to a particular AI (anastrozole, letrozole, or exemestane) or tamoxifen are distinct from each other, indicating that these mechanisms can be quite complex. Furthermore, we will describe two novel de novo AI-resistant cell lines that were generated from our laboratory. Initial characterization of these cells reveals that they are distinct from our acquired AI-resistant cell models. In addition, we will review potential therapies which may be useful for overcoming resistant breast cancers through studies using endocrine resistant cell lines. Finally, we will discuss the benefits and shortcomings of cell models. Together, the information presented in this review will provide us a better understanding of acquired and de novo resistance to tamoxifen and AI therapies, the use of appropriate cell models to better study these types of breast cancer, which are valuable for identifying novel treatments and strategies for overcoming both tamoxifen and AI-resistant breast cancers.
Models; endocrine resistance; tamoxifen; aromatase inhibitors
Progesterone is being utilized as a therapeutic means to ameliorate neuron loss and cognitive dysfunction following traumatic brain injury Although there have been numerous attempts to determine the means by which progesterone exerts neuroprotective effects, studies describing the underlying molecular mechanisms are lacking What has become clear, however, is the notion that progesterone can thwart several physiological processes that are detrimental to neuron function and survival, including inflammation, edema, demyelination and excitotoxicity One clue regarding the means by which progesterone has restorative value comes from the notion that these aforementioned biological processes all share the common theme of eliciting pronounced increases in intracellular calcium. Thus, we propose the hypothesis that progesterone regulation of calcium signaling underlies its ability to mitigate these cellular insults, ultimately leading to neuroprotection. Further, we describe recent findings that indicate neuroprotection is achieved via progesterone block of voltage-gated calcium channels, although additional outcomes may arise from blockade of various other ion channels and neurotransmitter receptors.
Progesterone; Brain Injury; Neuroprotection; Ischemia; Excitotoxicity; Calcium
Recent experiments from our laboratory are consistent with the idea that hypothalamic astrocytes are critical components of the central nervous system (CNS) mediated estrogen positive feedback mechanism. The “astrocrine hypothesis” maintains that ovarian estradiol rapidly increases free cytoplasmic calcium concentrations ([Ca2+]i) that facilitate progesterone synthesis in astrocytes. This hypothalamic neuroprogesterone along with the elevated estrogen from the ovaries allows for the surge release of gonadotropin-releasing hormone (GnRH) that triggers the pituitary luteinizing hormone (LH) surge. A narrow range of estradiol stimulated progesterone production supports an “off-on-off” mechanism regulating the transition from estrogen negative feedback to estrogen positive feedback, and back again. The rapidity of the [Ca2+]i response and progesterone synthesis support a non-genomic, membrane-initiated signaling mechanism. In hypothalamic astrocytes, membrane-associated estrogen receptors (mERs) signal through transactivation of the metabotropic glutamate receptor type 1a (mGluR1a), implying that astrocytic function is influenced by surrounding glutamatergic nerve terminals. Although other putative mERs, such as mERβ, STX-activated mER-Gαq, and G protein-coupled receptor 30 (GPR30), are present and participate in membrane-mediated signaling, their influence in reproduction is still obscure since female reproduction be it estrogen positive feedback or lordosis behavior requires mERα. The astrocrine hypothesis is also consistent with the well-known sexual dimorphism of estrogen positive feedback. In rodents, only post-pubertal females exhibit this positive feedback. Hypothalamic astrocytes cultured from females, but not males, responded to estradiol by increasing progesterone synthesis. Estrogen autoregulates its own signaling by regulating levels of mERα in the plasma membrane of female astrocytes. In male astrocytes, the estradiol-induced increase in mERα was attenuated, suggesting that membrane-initiated estradiol signaling (MIES) would also be blunted. Indeed, estradiol induced [Ca2+]i release in male astrocytes, but not to levels required to stimulate progesterone synthesis. Investigation of this sexual differentiation was performed using hypothalamic astrocytes from post-pubertal four core genotype (FCG) mice. In this model, genetic sex is uncoupled from gonadal sex. We demonstrated that animals that developed testes (XYM and XXM) lacked estrogen positive feedback, strongly suggesting that the sexual differentiation of progesterone synthesis is driven by the sex steroid environment during early development.
Estrogen; Progesterone; LH surge; Positive feedback; Estrogen receptor; Astrocyte
Glucocorticoids (GCs) are effective therapeutics commonly used in multiple myeloma (MM) treatment. Clarifying the pathway of GC-induced apoptosis is crucial to understanding the process of drug resistance and to the development of new targets for MM treatment. We have previously published results of a micro-array identifying glucocorticoid-induced leucine zipper (GILZ) as GC-regulated gene in MM.1S cells. Consistent with those results, GCs increased GILZ in MM cell lines and patient samples. Reducing the levels of GILZ with siRNA decreased GC-induced cell death suggesting GILZ may mediate GC-killing. We conducted a screen to identify other pathways that affect GILZ regulation and report that inhibitors of PI3-kinase/AKT enhanced GILZ expression in MM cell lines and clinical samples. The combination of dexamethasone (Dex) and LY294002, wortmannin, triciribine, or AKT inhibitor VIII dramatically up regulated GILZ levels and enhanced apoptosis. Addition of interleukin-6 (IL-6) or insulin-like growth factor (IGF1), both which activate the PI3-kinase/AKT pathway and inhibit GC killing, blocked up regulation of GILZ by GC and PI3-kinase/AKT inhibitors. In summary, these results identify GILZ as a mediator of GC killing, indicate a role of PI3-kinase/AKT in controlling GILZ regulation and suggest that the combination of PI3-kinase/AKT inhibitors and GCs may be a beneficial MM treatment.
Multiple myeloma; Glucocorticoid; Glucocorticoid receptor; GILZ; PI3-kinase; AKT; Signal transduction
The steroid hormone 1α,25-dihydroxyvitamin D3 [1α,25-(OH)2D3] is the most active metabolite of vitamin D3 which exerts its control over a multitude of biological processes related to calcium and phosphorus homeostasis, cell proliferation and differentiation, and immune regulation. Unfortunately, the therapeutic application of 1α,25-(OH)2D3 is limited by induction of hypercalcemia. The need for vitamin D compounds with selective biological profiles has stimulated the synthesis of more than three thousand analogs of 1α,25-(OH)2D3. Most of these compounds have structural modifications in the side chain and A-ring; there is also an increasing number of modifications in the CD-rings and limited number in the triene system (seco-B ring). Herein, we report the synthesis and biological evaluation of seco-A-19-nor analogs of 1α,25-dihydroxyvitamin D3, developed to study the role of ring A in the biological activity of 1α,25-(OH)2D3. Interestingly, compounds 2 and 4 show substantial ability to bind the vitamin D receptor and the former is also characterized by selective intestinal calcium transport activity.
Vitamin D analogs; 19-Norvitamin D3; Vitamin D receptor; VDR binding; Ring-A-seco analogs
► High dietary vitamin D was able to prevent premalignant lesions caused by AOM/DSS. ► Increasing vitamin D intake raised serum 25-D3 levels reaching a plateau ≥1000 IU/kg. ► Serum 25-D3 levels over 30 ng/ml are needed to prevent tumorigenesis.
Colorectal cancer (CRC) is one of the leading causes of cancer morbidity and mortality in Western countries. One of the risk factors for colorectal tumorigenesis is vitamin D insufficiency.
The aim of this study was to establish whether increasing dietary vitamin D intake can prevent or delay development of chemically induced preneoplastic lesions in the colon of mice.
We fed six weeks old female C57BL/6 J mice (n = 28) with increasing vitamin D3 concentrations (100, 400, 1000, 2500, 5000 IU/kg diet). To induce dysplasia, a preneoplastic lesion, we injected mice with the carcinogen azoxymethane (10 mg/kg) intraperitoneally, followed by three cycles of 2% dextran sodium sulfate salt, a tumor promoter, in the drinking water.
To test our hypothesis that high vitamin D intake prevents formation of preneoplastic lesions, we have investigated the effect of increasing dietary vitamin D on development of premalignant colorectal lesions, serum 25-hydroxyvitamin D3 (25-D3) levels, and expression of renal vitamin D system genes.
Dietary vitamin D concentration correlated inversely with dysplasia score (Spearman's correlation coefficient, ρ: −0.579, p = 0.002) and positively with serum 25-D3 levels (ρ: 0.752, p = 0.001). Increasing dietary vitamin D concentration beyond 1000 IU/kg led to no further increase in circulating 25-D3 levels, while the dysplasia score leveled out at ≥2500 IU/kg vitamin D. High dietary vitamin D intake led to increased renal mRNA expression of the vitamin D catabolizing enzyme cyp24a1 (ρ: 0.518, p = 0.005) and decreased expression of the vitamin D activating enzyme cyp27b1 (ρ: −0.452, p = 0.016), protecting the body from toxic serum levels of the active vitamin D metabolite 1,25-dihydroxyvitamin D3 (1,25-D3).
Our data showed that increasing dietary vitamin D intake is able to prevent chemically induced preneoplastic lesions. The maximum impact was achieved when the mice consumed more than 2500 IU vitamin D/kg diet.
This article is part of a Special Issue entitled ‘Vitamin D Workshop’.
Vitamin D; Colorectal cancer; Dysplasia; Chemoprevention; cyp24a1; cyp27b1
► Basal and calcitriol-induced expression of CYP24A1 is cell line dependent. ► Histone deacetylase and methyltransferase inhibitors increase CYP24A1 expression. ► Cell line specific drug response does not correlate with promoter methylation. ► Drug treatment may activate genes upstream of CYP24A1 and cause indirect induction.
Calcitriol is the hormonally active form of vitamin D and has anti-proliferative and pro-apoptotic effects. Calcitriol and its precursor calcidiol (25(OH)D3) are degraded by the 1,25-dihydroxyvitamin D3 24-hydroxylase (CYP24A1). This enzyme is overexpressed in colorectal tumors, however, the mechanisms of this overexpression remain to be elucidated. CYP24A1 mRNA level differs among colorectal cancer cell lines and range from almost undetectable to high. Since DNA methylation and histone acetylation regulate CYP24A1 gene expression in prostate cancer cell lines, we investigated whether epigenetic mechanisms could explain the differences in basal expression of CYP24A1 in colon cancer cells. Methyltransferase inhibitor 5-aza-2′-deoxycytidine (DAC) treatment resulted in an over 50-fold induction of CYP24A1 mRNA expression in Coga1A and HT-29 cells but in no response in Caco2/AQ and Coga13 cells. This finding is supported by a strong increase in CYP24A1 activity after DAC treatment in Coga1A (35%). In addition, calcitriol and DAC had synergistic effects on CYP24A1 gene transcription. Interestingly, the CYP24A1 promoter was not methylated in Coga1A and HT-29 (<5%), while in Caco2/AQ it was 62% methylated. This suggests that DNA demethylation must activate genes upstream of CYP24A1 rather than act on the gene itself. However, transcriptional regulators of CYP24A1 such as vitamin D receptor (VDR), retinoid X receptor (RXR), specificity protein 1 (SP1), or mediator complex subunit 1 (MED1) were not upregulated. We conclude that in colon cancer cells, CYP24A1 gene expression is inducible by methyltransferase and some histone deacetylase inhibitors in a cell line-dependent manner. This effect does not correlate with the methylation state of the promoter and therefore must affect genes upstream of CYP24A1.
This article is part of a Special Issue ‘Vitamin D Workshop’.
SP1, specificity protein 1; MED1, mediator complex subunit 1; DAC, 5-aza-2′-deoxycytidine; TSA, trichostatin A; CYP24A1; Histone deacetylase inhibitors; Methylation; Epigenetics; 5-Aza-2′-deoxycytidine; Trichostatin A
Type 5 17β-hydroxysteroid dehydrogenase (AKR1C3) is the major enzyme in the prostate that reduces 4-androstene-3,17-dione (Δ4-Adione) to the androgen receptor (AR) ligand testosterone. AKR1C3 is upregulated in prostate cancer (PCa) and castrate resistant prostate cancer (CRPC) that develops after androgen deprivation therapy. PCa and CRPC often depend on intratumoral androgen biosynthesis and upregulation of AKR1C3 could contribute to intracellular synthesis of AR ligands and stimulation of proliferation through AR signalling. To test this hypothesis, we developed an LNCaP prostate cancer cell line overexpressing AKR1C3 (LNCaP-AKR1C3) and compared its metabolic and proliferative responses to Δ4-Adione treatment with that of the parental, AKR1C3 negative LNCaP cells. In LNCaP and LNCaP-AKR1C3 cells, metabolism proceeded via 5α-reduction to form 5α-androstane-3,17-dione and then (epi)androsterone-3-glucuronide. LNCaP-AKR1C3 cells made significantly higher amounts of testosterone-17β-glucuronide. When 5α-reductase was inhibited by finasteride, the production of testosterone-17β-glucuronide was further elevated in LNCaP-AKR1C3 cells. When AKR1C3 activity was inhibited with indomethacin the production of testosterone-17β-glucuronide was significantly decreased. Δ4-Adione treatment stimulated cell proliferation in both cell lines. Finasteride inhibited LNCaP cell proliferation, consistent with 5α-androstane-3,17-dione acting as the major metabolite that stimulates growth by binding to the mutated AR. However, LNCaP-AKR1C3 cells were resistant to the growth inhibitory properties of finasteride, consistent with the diversion of Δ4-Adione metabolism from 5α-reduced androgens to increased formation of testosterone. Indomethacin did not result in differences in Δ4-Adione induced proliferation since this treatment led to the same metabolic profile in LNCaP and LNCaP-AKR1C3 cells. We conclude that AKR1C3 overexpression diverts androgen metabolism to testosterone that results in proliferation in androgen sensitive prostate cancer. This effect is seen despite high levels of uridine glucuronosyl transferases suggesting that AKR1C3 activity can surmount the effects of this elimination pathway. Treatment options in prostate cancer that target 5α-reductase where AKR1C3 co-exists may be less effective due to the diversion of Δ4-Adione to testosterone.
type 5 17β-hydroxysteroid dehydrogenase; 4-androstene-3; 17-dione; 5α-reductase; indomethacin; castrate resistant prostate cancer
Human ketosteroid reductases of the aldo-keto reductase (AKR) superfamily, i.e. AKR1C1-4, are implicated in the biotransformation of synthetic steroid hormones. Norethynodrel (NOR, 17α-ethynyl-17β-hydroxy-estra-5(10)-en-3-one), the progestin component of the first marketed oral contraceptive, is known to undergo rapid and extensive metabolism to 3α- and 3β-hydroxy metabolites. The ability of the four human AKR1C enzymes to catalyze the metabolism of NOR has now been characterized. AKR1C1 and AKR1C2 almost exclusively converted NOR to 3β-hydroxy NOR, while AKR1C3 gave 3β-hydroxy NOR as the main product and AKR1C4 predominantly formed 3α-hydroxy NOR. Individual AKR1C enzymes also displayed distinct kinetic properties in the reaction of NOR. In contrast, norethindrone (NET), the Δ4-isomer of NOR and the most commonly used synthetic progestin, was not a substrate for the AKR1C enzymes. NOR is also structurally identical to the hormone replacement therapeutic tibolone (TIB), except TIB has a methyl group at the 7α-position. Product profiles and kinetic parameters for the reduction of NOR catalyzed by each individual AKR1C isoform were identical to those for the reduction of TIB catalyzed by the respective isoform. These data suggest that the presence of the 7α-methyl group has a minimal effect on the stereochemical outcome of the reaction and kinetic behavior of each enzyme. Results indicate a role of AKR1C in the hepatic and peripheral metabolism of NOR to 3α- and 3β-hydroxy NOR and provide insights into the differential pharmacological properties of NOR, NET and TIB.
Ketosteroid reduction; progestin; oral contraceptive
CYP27A1 is a mitochondrial cytochrome P450 which can hydroxylate vitamin D3 and cholesterol at carbons 25 and 26, respectively. The product of vitamin D3 metabolism, 25-hydroxyvitamin D3, is the precursor to the biologically active hormone, 1α,25-dihydroxyvitamin D3. CYP27A1 is attached to the inner mitochondrial membrane and substrates appear to reach the active site through the membrane phase. We have therefore examined the ability of bacterially expressed and purified CYP27A1 to metabolize substrates incorporated into phospholipid vesicles which resemble the inner mitochondrial membrane. We also examined the ability of CYP27A1 to metabolize 20-hydroxyvitamin D3 (20(OH)D3), a novel non-calcemic form of vitamin D derived from CYP11A1 action on vitamin D3 which has anti-proliferative activity on keratinocytes, leukemic and myeloid cells. CYP27A1 displayed high catalytic activity towards cholesterol with a turnover number (kcat) of 9.8 min−1 and Km of 0.49 mol/mol phospholipid (510 μM phospholipid). The Km value of vitamin D3 was similar for that of cholesterol, but the kcat was 4.5-fold lower. 20(OH)D3 was metabolized by CYP27A1 to two major products with a kcat/Km that was 2.5-fold higher than that for vitamin D3, suggesting that 20(OH)D3 could effectively compete with vitamin D3 for catalysis. NMR and mass spectrometric analyses revealed that the two major products were 20,25-dihydroxyvitamin D3 and 20,26-dihydroxyvitamin D3, in almost equal proportions. Thus the presence of the 20-hydroxyl group on the vitamin D3 side chain enables it to be metabolized more efficiently than vitamin D3, with carbon 26 in addition to carbon 25 becoming a major site of hydroxylation. Our study reports the highest kcat for the 25-hydroxylation of vitamin D3 by any human cytochrome P450 suggesting that CYP27A1 might be an important contributor to the synthesis of 25-hydroxyvitamin D3, particularly in tissues where it is highly expressed.
CYP27A1; vitamin D; cholesterol; cytochrome P450; phospholipid vesicles
The reactive stromal phenotype is an important factor for prostate cancer progression and may be a new target for treatment and prevention. A new high efficiency preclinical protocol, the EPI bioassay, reflects the interaction of endocrine, paracrine and immune, (EPI) factors on induced androgen metabolism in human prostate reactive stroma. The bioassay is based on co-culturing human primary prostate stromal cells and LAPC-4 prostatic adenocarcinoma cells in a downscaled format of 96-well-plates for testing multiple doses of multiple target compounds. Metabolism of dehydroepiandrosterone (DHEA) with or without TGFβ1–induced stimulation (D+T) of the reactive stroma phenotype was assessed by increased testosterone in the media and PSA production of the epithelial prostate cancer cells. By using the non-metabolizable androgen R1881, effects from direct androgen action were distinguished from stromal androgen production from DHEA. Stromal cell androgenic bioactivity was confirmed using conditioned media from D+T-treated stromal cell monocultures in an androgen-inducible AR screening assay. We further showed that both agonists to estrogen receptor (ER), DPN (ERβ) and PPT (ERα), as well as estrogenic natural compounds including soy isoflavones attenuated D+T-induced PSA production. Studies with the pure ER agonists showed that activating either ERα or ERβ could inhibit both D+T-mediated and R1881-mediated PSA production with the D+T effect being more pronounced. In conclusion, natural compounds with estrogenic activity and pure ER agonists are very potent inhibitors of stromal conversion of DHEA to androgenic metabolites. More studies are needed to characterize the mechanisms involved in estrogenic modulation of the endocrine-immune-paracrine balance of the prostate microenvironment.
Uterine leiomyomas are highly prevalent and often symptomatic. Current medical therapies are limited. A novel, potent, selective, orally active therapy is needed.
Objective and Methods
To determine the progesterone receptor (PR) specificity and activation, endometrial response, and impact on proliferation and extracellular matrix (ECM) production of the novel non-steroidal selective progesterone receptor modulators (SPRMs) CP8863 and CP8947 in human immortalized leiomyoma and patient-matched myometrial cells. Receptor binding in vitro was assessed using LNCaP, Ishikawa, T-47D, and HeLa cell extracts for AR, ER-α, PR, and GR, respectively. Progestational activity assessed by alkaline phosphatase assay in T47D cells and ER-α expression in human leiomyoma and myometrial cells. In vivo progestational activity assayed by the McPhail assay. Proliferation and gene expression studies (q RT-PCR and western blot) were performed in immortalized leiomyoma and myometrial cells.
Both CP8863 and CP8947 is highly selective for PR but not for ER-α, AR, and GR. Both induced alkaline phosphatase comparably to progesterone, while CP8947 induced ER-α in leiomyoma cells but not myometrial cells. CP8947 was progestational in rabbit endometrium. Nanomolar CP8947 treatment inhibited human leiomyoma but not myometrial cell proliferation. The decreased proliferation correlated with increased TRAIL and caspase -7, suggesting induction of apoptosis in leiomyoma cells. ECM components were decreased in leiomyoma cells, including COL1A1 and COL7A1 at nanomolar concentrations.
CP8947 was a potent novel non-steroidal SPRM that was selective for PR, showed progestational activity in endometrium, inhibited leiomyoma cell proliferation (potentially via induction of apoptosis), and decreased ECM component production, without disrupting myometrial cell proliferation.
Leiomyoma; selective progesterone receptor modulator; endometrium; apoptosis; myometrium; extracellular matrix
The human steroidogenic cytochromes P450 CYP17A1 (P450c17, 17α-hydroxylase/17,20-lyase) and CYP21A2 (P450c21, 21-hydroxylase) are required for the biosynthesis of androgens, glucocorticoids, and mineralocorticoids. Both enzymes hydroxylate progesterone at adjacent, distal carbon atoms and show limited tolerance for substrate modification. Halogenated substrate analogs have been employed for many years to probe cytochrome P450 catalysis and to block sites of reactivity, particularly for potential drugs. Consequently, we developed efficient synthetic approaches to introducing one or more halogen atom to the 17- and 21-positions of progesterone and pregnenolone. In particular, novel 21,21,21-tribromoprogesterone and 21,21,21-trichloroprogesterone were synthesized using the nucleophilic addition of either bromoform or chloroform anion onto an aldehyde precursor as the key step to introduce the trihalomethyl moieties. When incubated with microsomes from yeast expressing human CYP21A2 or CYP17A1 with P450-oxidoreductase, CYP21A2 metabolized 17-fluoroprogesterone to a single product, whereas incubations with CYP17A1 gave no products. Halogenated steroids provide a robust system for exploring the substrate tolerance and catalytic plasticity of human steroid hydroxylases.
The present study investigated the association between variants in the vitamin D receptor gene (VDR) and protein tyrosine phosphatase, non-receptor type 2 gene (PTPN2), as well as an interaction between VDR and PTPN2 and the risk of islet autoimmunity (IA) and progression to type 1 diabetes (T1D). The Diabetes Autoimmunity Study in the Young (DAISY) has followed children at increased risk of T1D since 1993. Of the 1692 DAISY children genotyped for VDR rs1544410, VDR rs2228570, VDR rs11568820, PTPN2 rs1893217, and PTPN2 rs478582, 111 developed IA, defined as positivity for GAD, insulin or IA-2 autoantibodies on 2 or more consecutive visits, and 38 IA positive children progressed to T1D. Proportional hazards regression analyses were conducted.
There was no association between IA development and any of the gene variants, nor was there evidence of a VDR*PTPN2 interaction. Progression to T1D in IA positive children was associated with the VDR rs2228570 GG genotype (HR: 0.49, 95% CI: 0.26–0.92) and there was an interaction between VDR rs1544410 and PTPN2 rs1893217 (pinteraction = 0.02). In children with the PTPN2 rs1893217 AA genotype, the VDR rs1544410 AA/AG genotype was associated with a decreased risk of T1D (HR: 0.24, 95% CI: 0.11–0.53, p = 0.0004), while in children with the PTPN2 rs1893217 GG/GA genotype, the VDR rs1544410 AA/AG genotype was not associated with T1D (HR: 1.32, 95% CI: 0.43–4.06, p = 0.62). These findings should be replicated in larger cohorts for confirmation. The interaction between VDR and PTPN2 polymorphisms in the risk of progression to T1D offers insight concerning the role of vitamin D in the etiology of T1D.
Vitamin D; Genetics; Epidemiology; Islet autoimmunity; Type 1 diabetes; Vitamin D receptor
The current study was designed to examine the sulfation of bile acids and bile alcohols by the Zebra danio (Danio rerio) SULTs in comparison with human SULTs. A systematic analysis using the fifteen Zebra danio SULTs revealed that SULT3 ST2 and SULT3 ST3 were the major bile acid/alcohol-sulfating SULTs. Among the eleven human SULTs, only SULT2A1 was found to be capable of sulfating bile acids and bile alcohols. To further investigate the sulfation of bile acids and bile alcohols by the two Zebra danio SULT3 STs and the human SULT2A1, pH-dependence and kinetics of the sulfation of bile acids/alcohols were analyzed. pH-dependence experiments showed that the mechanisms underlying substrate recognition for the sulfation of lithocholic acid (a bile acid) and 5α-petromyzonol (a bile alcohol) differed between the human SULT2A1 and the Zebra danio SULT3 ST2 and ST3. Kinetic analysis indicated that both the two Zebra danio SULT3 STs preferred petromyzonol as substrate compared to bile acids. In contrast, the human SULT2A1 was more catalytically efficient toward lithocholic acid than petromyzonol. Collectively, the results imply that the Zebra danio and human SULTs have evolved to serve for the sulfation of, respectively, bile alcohols and bile acids, matching the cholanoid profile in these two vertebrate species.
Sulfotransferase; Sulfation; Zebra danio; Bile acid
7-Dehydrocholesterol reductase (DHCR7) catalyzes the final step in cholesterol synthesis. The enzyme utilizes NADPH as a source of electrons and has been reported to require NADPH-cytochrome P450 reductase (POR) as its redox partner. To test this hypothesis, microsomes were prepared from the livers of mice in which hepatic cytochrome P450 reductase expression was extinguished during maturation. These microsomes contained negligible levels of POR but had 2.5-fold greater DHCR7 activity than did microsomes from wild-type mice. Consistent with this greater activity, immunoblot analysis of DHCR7 expression indicated that DHCR7 protein levels were elevated two-fold in POR-null microsomes. Addition of POR to these microsomes provided no stimulation of DHCR7 activity, confirming the lack of a role for POR in DHCR7 activity. Because the original observation that POR was necessary for DHCR7 activity was based, in part, on antibody inhibition studies with POR antibody, the ability of an antibody to the full-length POR protein to inhibit DHCR7 activity and cytochrome c reductase activity was tested; the antibody had no effect on DHCR7 activity but decreased cytochrome c reductase activity (a POR-catalyzed reaction) by 50%. Immunoblot analysis further demonstrated no cross-reactivity between POR and DHCR7 with antibodies to either protein. We conclude that cytochrome P450 reductase is not involved in 7-dehydrocholesterol reductase activity.
7-Dehydrocholesterol reductase; NADPH-cytochrome P450 reductase; cholesterol synthesis; Smith-Lemli-Opitz Syndrome; liver microsomes
Accumulating evidence supports the theory that breast cancer arises from a subpopulation of mammary stem/progenitor cell which posses the ability to self-renew. However, the involvement of estrogen signaling in regulation of breast cancer stem/progenitor cells has not been fully established, mainly because expression and function of ER-α in breast cancer stem cells remains controversial. Previously, our laboratory cloned a variant of ER-α, ER-α36, and found that ER-α36-mediated non-genomic estrogen signaling plays an important role in malignant growth of triple-negative breast cancer cells. In this study, we found that ER-α36 was highly expressed in ER-negative breast cancer SK-BR-3 cells and mediated non-genomic estrogen signaling such as activation of the MAPK/ERK signaling in these cells. Knock-down of ER-α36 expression in these cells using the shRNA method diminished their responsiveness to estrogen and significantly down-regulated HER2 expression. HER2 signaling activated ER-α36 transcription through an AP1 site in the ER-α36 promoter and ER-α36 physically interacted with HER2. We also found that ER-α36 is highly expressed in a subset of SK-BR-3 cells that was positive for ALDH1, a breast cancer stem cell marker, and knock-down of ER-α36 expression reduced the population of ALDH1 positive cells. Our results thus demonstrated that ER-α36 positively regulates HER2 expression and the population of ALDH1 positive breast cancer cells, and suggested that non-genomic estrogen signaling mediated by ER-α36 is involved in maintenance and regulation of breast cancer stem cells.
HER2; ER-α36; ALDH1; Breast cancer stem cells
Environmental chemicals have significant impacts on biological systems. Chemical exposures during early stages of development can disrupt normal patterns of development and thus dramatically alter disease susceptibility later in life. Endocrine disrupting chemicals (EDCs) interfere with the body's endocrine system and produce adverse developmental, reproductive, neurological, cardiovascular, metabolic and immune effects in humans. A wide range of substances, both natural and man-made, are thought to cause endocrine disruption, including pharmaceuticals, dioxin and dioxin-like compounds, polychlorinated biphenyls, DDT and other pesticides, and components of plastics such as bisphenol A (BPA) and phthalates. EDCs are found in many everyday products– including plastic bottles, metal food cans, detergents, flame retardants, food additives, toys, cosmetics, and pesticides. EDCs interfere with the synthesis, secretion, transport, activity, or elimination of natural hormones. This interference can block or mimic hormone action, causing a wide range of effects. This review focuses on the mechanisms and modes of action by which EDCs alter hormone signaling. It also includes brief overviews of select disease endpoints associated with endocrine disruption.
GPER/GPR30 is a seven-transmembrane G protein-coupled estrogen receptor that regulates many aspects of mammalian biology and physiology. We have previously described both a GPER-selective agonist G-1 and antagonist G15 based on a tetrahydro-3H-cyclopenta[c]quinoline scaffold. The antagonist lacks an ethanone moiety that likely forms important hydrogen bonds involved in receptor activation. Computational docking studies suggested that the lack of the ethanone substituent in G15 could minimize key steric conflicts, present in G-1, that limit binding within the ERα ligand binding pocket. In this report, we identify low-affinity cross-reactivity of the GPER antagonist G15 to the classical estrogen receptor ERα. To generate an antagonist with enhanced selectivity, we therefore synthesized an isosteric G-1 derivative, G36, containing an isopropyl moiety in place of the ethanone moiety. We demonstrate that G36 shows decreased binding and activation of ERα, while maintaining its antagonist profile towards GPER. G36 selectively inhibits estrogen-mediated activation of PI3K by GPER but not ERα. It also inhibits estrogen- and G-1-mediated calcium mobilization as well as ERK1/2 activation, with no effect on EGF-mediated ERK1/2 activation. Similar to G15, G36 inhibits estrogen- and G-1-stimulated proliferation of uterine epithelial cells in vivo. The identification of G36 as a GPER antagonist with improved ER counterselectivity represents a significant step towards the development of new highly selective therapeutics for cancer and other diseases.