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Sample records for aromatase inhibitor resistance

  1. Understanding the mechanisms of aromatase inhibitor resistance

    PubMed Central

    2012-01-01

    Aromatase inhibitors (AIs) have a central role in the treatment of breast cancer; however, resistance is a major obstacle to optimal management. Evidence from endocrine, molecular and pathological measurements in clinical material taken before and after therapy with AIs and data from clinical trials in which AIs have been given as treatment either alone or in combination with other targeted agents suggest diverse causes for resistance. These include inherent tumour insensitivity to oestrogen, ineffective inhibition of aromatase, sources of oestrogenic hormones independent of aromatase, activation of signalling by non-endocrine pathways, enhanced cell survival and selection of hormone-insensitive cellular clones during treatment. PMID:22277572

  2. NEW EXPERIMENTAL MODELS FOR AROMATASE INHIBITOR RESISTANCE

    PubMed Central

    Chen, Shiuan; Masri, Selma; Hong, Yanyan; Wang, Xin; Phung, Sheryl; Yuan, Yate-Ching; Wu, Xiwei

    2009-01-01

    Clinical trials have demonstrated the importance of aromatase inhibitor (AI) therapy in the effective treatment of hormone-dependent breast cancers. In contrast to tamoxifen, an antagonist of the estrogen receptor (ER), AIs have shown to be better tolerated along with decreased recurrence rates of the disease. Currently, three third-generation AIs are being used: exemestane, letrozole and anastrozole. Our laboratory is attempting to understand several aspects of aromatase inhibitor functionality. In this paper, we first review recent findings from our structure-function studies of aromatase as well as the molecular characterization of the interaction between AIs and aromatase. Based on these studies, we propose new evidence for the interaction of letrozole and exemestane with aromatase. In addition, we will discuss recent results generated from our AI-resistant cell lines. Our laboratory has generated MCF-7aro cells that are resistant to letrozole, anastrozole, exemestane and tamoxifen. Basic functional characterization of aromatase and ERα in these resistant cell lines has been done and microarray analysis has been employed in order to better understand the mechanism responsible for AI resistance on a genome-wide scale. The results generated so far suggest the presence of at least four types of resistant cell lines. Overall, the information presented in this paper supplements our understanding of AI function, and such information can be valuable for the development of treatment strategies against AI resistant breast cancers. PMID:17611102

  3. What do we know about the mechanisms of aromatase inhibitor resistance?

    PubMed Central

    Chen, Shiuan; Masri, Selma; Wang, Xin; Phung, Sheryl; Yuan, Yate-Ching; Wu, Xiwei

    2007-01-01

    Clinical trials have demonstrated the importance of aromatase inhibitor (AI) therapy in the effective treatment of hormone-dependent breast cancers. Yet, as with all prolonged drug therapy, resistance to aromatase inhibitors does develop. To date, the precise mechanism responsible for resistance to aromatase inhibitors is not completely understood. In this paper, several mechanisms of de novo/intrinsic resistance and acquired resistance to AIs are discussed. These mechanisms are hypothesized based on important findings from a number of laboratories. To better understand this question, our lab has generated, in vitro, breast cancer cell lines that are resistant to aromatase inhibitors. Resistant cell lines were generated over a prolonged period of time using the MCF-7aro (aromatase overexpressed) breast cancer line. These cell lines are resistant to the aromatase inhibitors letrozole, anastrozole and exemestane and the anti-estrogen tamoxifen, for comparison. Two types of resistant cell lines have been generated, those that grow in the presence of Testosterone (T) which is needed for cell growth, and resistant lines that are cultured in the presence of inhibitor only (no T). In addition to functional characterization of aromatase and ERα in these resistant cell lines, microarray analysis has been employed in order to determine differential gene expression within the aromatase inhibitor resistant cell lines versus tamoxifen, in order to better understand the mechanism responsible for AI resistance on a genome-wide scale. We anticipate that our studies will generate important information on the mechanisms of AI resistance. Such information can be valuable for the development of treatment strategies against AI resistant breast cancers. PMID:17055257

  4. Aromatase, Aromatase Inhibitors, and Breast Cancer

    PubMed Central

    Chumsri, Saranya; Howes, Timothy; Bao, Ting; Sabnis, Gauri; Brodie, Angela

    2011-01-01

    Estrogens are known to be important in the growth of breast cancers in both pre- and postmenopausal women. As the number of breast cancer patients increases with age, the majority of breast cancer patients are postmenopausal women. Although estrogens are no longer made in the ovaries after menopause, peripheral tissues produce sufficient concentrations to stimulate tumor growth. As aromatase catalyzes the final and rate-limiting step in the biosynthesis of estrogen, inhibitors of this enzyme are effective targeted therapy for breast cancer. Three aromatase inhibitors (AIs) are now FDA approved and have been shown to be more effective than the antiestrogen tamoxifen and are well tolerated. AIs are now a standard treatment for postmenopausal patients. AIs are effective in adjuvant and first-line metastatic setting. This review describes the development of AIs and their current use in breast cancer. Recent research focuses on elucidating mechanisms of acquired resistance that may develop in some patients with long term AI treatment and also on innate resistance. Preclinical data in resistance models demonstrated that the crosstalk between ER and other signaling pathways particularly MAPK and PI3K/Akt is an important resistant mechanism. Blockade of these other signaling pathways is an attractive strategy to circumvent the resistance to AI therapy in breast cancer. Several clinical trials are ongoing to evaluate the role of these novel targeted therapies to reverse resistance to AIs. PMID:21335088

  5. Sequencing of aromatase inhibitors

    PubMed Central

    Bertelli, G

    2005-01-01

    Since the development of the third-generation aromatase inhibitors (AIs), anastrozole, letrozole and exemestane, these agents have been the subject of intensive research to determine their optimal use in advanced breast cancer. Not only have they replaced progestins in second-line therapy and challenged the role of tamoxifen in first-line, but there is also evidence for a lack of cross-resistance between the steroidal and nonsteroidal AIs, meaning that they may be used in sequence to obtain prolonged clinical benefit. Many questions remain, however, as to the best sequence of the two types of AIs and of the other available agents, including tamoxifen and fulvestrant, in different patient groups. PMID:16100523

  6. GDNF-RET signaling in ER-positive breast cancers is a key determinant of response and resistance to aromatase inhibitors.

    PubMed

    Morandi, Andrea; Martin, Lesley-Ann; Gao, Qiong; Pancholi, Sunil; Mackay, Alan; Robertson, David; Zvelebil, Marketa; Dowsett, Mitch; Plaza-Menacho, Ivan; Isacke, Clare M

    2013-06-15

    Most breast cancers at diagnosis are estrogen receptor-positive (ER(+)) and depend on estrogen for growth and survival. Blocking estrogen biosynthesis by aromatase inhibitors has therefore become a first-line endocrine therapy for postmenopausal women with ER(+) breast cancers. Despite providing substantial improvements in patient outcome, aromatase inhibitor resistance remains a major clinical challenge. The receptor tyrosine kinase, RET, and its coreceptor, GFRα1, are upregulated in a subset of ER(+) breast cancers, and the RET ligand, glial-derived neurotrophic factor (GDNF) is upregulated by inflammatory cytokines. Here, we report the findings of a multidisciplinary strategy to address the impact of GDNF-RET signaling in the response to aromatase inhibitor treatment. In breast cancer cells in two-dimensional and three-dimensional culture, GDNF-mediated RET signaling is enhanced in a model of aromatase inhibitor resistance. Furthermore, GDNF-RET signaling promoted the survival of aromatase inhibitor-resistant cells and elicited resistance in aromatase inhibitor-sensitive cells. Both these effects were selectively reverted by the RET kinase inhibitor, NVP-BBT594. Gene expression profiling in ER(+) cancers defined a proliferation-independent GDNF response signature that prognosed poor patient outcome and, more importantly, predicted poor response to aromatase inhibitor treatment with the development of resistance. We validated these findings by showing increased RET protein expression levels in an independent cohort of aromatase inhibitor-resistant patient specimens. Together, our results establish GDNF-RET signaling as a rational therapeutic target to combat or delay the onset of aromatase inhibitor resistance in breast cancer.

  7. Aromatase Inhibitors and Other Compounds for Lowering Breast Cancer Risk

    MedlinePlus

    ... Cancer Risk and Prevention Aromatase Inhibitors for Lowering Breast Cancer Risk Aromatase inhibitors (drugs that lower estrogen levels) ... day. Can aromatase inhibitors lower the risk of breast cancer? Aromatase inhibitors are used mainly to treat hormone ...

  8. AKT-aro and HER2-aro, models for de novo resistance to aromatase inhibitors; molecular characterization and inhibitor response studies.

    PubMed

    Wong, Cynthie; Wang, Xin; Smith, David; Reddy, Kaladhar; Chen, Shiuan

    2012-07-01

    Aromatase inhibitors (AI) are currently the first line therapy for estrogen receptor (ER)-positive postmenopausal women. De novo AI resistance is when a patient intrinsically does not respond to an AI therapy as well as other targeted endocrine therapy. To characterize this type of resistance and to examine potential therapies for treatment, we have generated two cell models for de novo resistance. These models derive from MCF-7 cells that stably overexpress aromatase and Akt (AKT-aro) or HER2 (HER2-aro). Evaluation of these cell lines revealed that the activities of aromatase and ER were inhibited by AI and ICI 187280 (ICI) treatment, respectively; however, cell growth was resistant to therapy. Proliferation in the presence of the pure anti-estrogen ICI, indicates that these cells do not require ER for cell growth and distinguishes these cells from the acquired AI resistant cells. We further determined that the HSP90 inhibitor 17-DMAG suppressed the growth of the AI-resistant cell lines studied. Our analysis revealed 17-DMAG-mediated decreased expression of growth promoting signaling proteins. It was found that de novo AI resistant AKT-aro and HER2-aro cells could not be resensitized to letrozole or ICI by treatment with 17-DMAG. In summary, we have generated two cell lines which display the characteristics of de novo AI resistance. Together, these data indicate the possibility that HSP90 inhibitors may be a viable therapy for endocrine therapy resistance although additional clinical evaluation is needed.

  9. Natural products as aromatase inhibitors.

    PubMed

    Balunas, Marcy J; Su, Bin; Brueggemeier, Robert W; Kinghorn, A Douglas

    2008-08-01

    With the clinical success of several synthetic aromatase inhibitors (AIs) in the treatment of postmenopausal estrogen receptor-positive breast cancer, researchers have also been investigating the potential of natural products as AIs. Natural products from terrestrial and marine organisms provide a chemically diverse array of compounds not always available through current synthetic chemistry techniques. Natural products that have been used traditionally for nutritional or medicinal purposes (e.g., botanical dietary supplements) may also afford AIs with reduced side effects. A thorough review of the literature regarding natural product extracts and secondary metabolites of plant, microbial, and marine origin that have been shown to exhibit aromatase inhibitory activity is presented herein.

  10. Substituted androstanes as aromatase inhibitors

    NASA Astrophysics Data System (ADS)

    Levina, Inna S.

    1998-11-01

    The synthesis and structure-activity relationships of inhibitors of steroid aromatase which catalyses the last stage of a multistep biotransformation of cholesterol into estrogens, viz., aromatisation of C19-steroids into C18-phenolic steroids, are discussed. Compounds of the androstane series which are structurally related to the natural substrate, viz., androst-4-ene-3,17-dione, are the subjects of consideration. The review encompasses problems of synthesis of various substituted androstanes and their aromatase-inhibiting activities and structural requirements for selective specific aromatase inhibitors based on in vitro and in vivo structure-activity studies of compounds synthesised, their biological properties and the results of clinical trials. Special attention is paid to practical applications of aromatase inhibitors in the treatment of hormone-dependent mammary and ovarian tumours as well as benign prostatic tumours. In writing this report, the author has used all the information currently available in the chemical, biochemical, endocrinological and medicinal literature as well as in patents. The bibliography includes 173 references.

  11. Profiles of miRNAs matched to biology in aromatase inhibitor resistant breast cancer

    PubMed Central

    Hoppe, Reiner; Fan, Ping; Büttner, Florian; Winter, Stefan; Tyagi, Amit K.; Cunliffe, Heather; Jordan, V. Craig; Brauch, Hiltrud

    2016-01-01

    Aromatase inhibitor (AI) resistance during breast cancer treatment is mimicked by MCF-7:5C (5C) and MCF-7:2A (2A) cell lines that grow spontaneously. Survival signaling is reconfigured but cells are vulnerable to estradiol (E2)-inducible apoptosis. These model systems have alterations of stress related pathways including the accumulation of endoplasmic reticulum, oxidative, and inflammatory stress that occur prior to E2-induced apoptosis. We investigated miRNA expression profiles of 5C and 2A to characterize their AI resistance phenotypes. Affymetrix GeneChip miRNA2.0 arrays identified 184 miRNAs differentially expressed in 2A and 5C compared to E2-free wild-type MCF-7:WS8. In 5C, 34 miRNAs of the DLK1-DIO3 locus and miR-31 were overexpressed, whereas miR-222 was low. TCGA data revealed poor and favorable overall survival for low miR-31 and miR-222 levels, respectively (HR=3.0, 95% CI:1.9-4.8; HR=0.3, 95% CI:0.1-0.6). Targets of deregulated miRNAs were identified using CLIP-confirmed TargetScan predictions. KEGG enrichment analyses for 5C- and 2A-specific target gene sets revealed pathways associated with cell proliferation including insulin, mTOR, and ErbB signaling as well as immune response and metabolism. Key genes overrepresented in 5C- and 2A-specific pathway interaction networks including EGFR, IGF1R and PIK3R1 had lower protein levels in 5C compared to 2A and were found to be differentially modulated by respective miRNA sets. Distinct up-regulated miRNAs from the DLK1-DIO3 locus may cause these attenuative effects as they are predicted to interact with corresponding 3′ untranslated regions. These new miRNA profiles become an important regulatory database to explore E2-induced apoptotic mechanisms of clinical relevance for the treatment of resistant breast cancer. PMID:27659519

  12. [Indoleamine 2,3-Dioxygenase Activity during Fulvestrant Therapy for Aromatase Inhibitor-Resistant Metastatic Breast Cancer].

    PubMed

    Sakurai, Kenichi; Fujisaki, Shigeru; Suzuki, Shuhei; Adachi, Keita; Nagashima, Saki; Masuo, Yuki; Tomita, Ryouichi; Gonda, Kenji; Enomoto, Katsuhisa; Amano, Sadao; Matsuo, Sadanori; Umeda, Nao

    2015-10-01

    We evaluated the clinical significance of indoleamine 2,3-dioxygenase (IDO) during fulvestrant therapy for aromatase inhibitor (AI)-resistant metastatic breast cancer. IDO activity can be measured by the tryptophan (Trp)/kynurenine (Kyn) ratio. Trp and Kyn were measured with high performance liquid chromatography (HPLC). Patients with AI resistant metastatic breast cancer had a 28.6% response rate to fulvestrant therapy, and the clinical benefit rate was 76.2%. AI-resistant metastatic breast cancer patients with distant metastases had a lower serum Trp/Kyn level than patients who had local recurrences. During fulvestrant therapy, IDO activity significantly decreased in the fulvestrant responder group compared to that in the fulvestrant non-responder group. During fulvestrant therapy, the IDO activity correlated with the number of metastatic lesions. These results suggest that measuring the Trp/Kyn ratio is useful for evaluating immunological metastatic status during endocrine therapy.

  13. Natural Products as Aromatase Inhibitors

    PubMed Central

    Balunas, Marcy J.; Su, Bin; Brueggemeier, Robert W.; Kinghorn, A. Douglas

    2010-01-01

    With the clinical success of several synthetic aromatase inhibitors (AIs) in the treatment of postmenopausal estrogen receptor-positive breast cancer, researchers have also been investigating also the potential of natural products as AIs. Natural products from terrestrial and marine organisms provide a chemically diverse array of compounds not always available through current synthetic chemistry techniques. Natural products that have been used traditionally for nutritional or medicinal purposes (e.g., botanical dietary supplements) may also afford AIs with reduced side effects. A thorough review of the literature regarding natural product extracts and secondary metabolites of plant, microbial, and marine origin that have been shown to exhibit aromatase inhibitory activity is presented herein. PMID:18690828

  14. Aurora kinase A and B as new treatment targets in aromatase inhibitor-resistant breast cancer cells.

    PubMed

    Hole, Stine; Pedersen, Astrid M; Lykkesfeldt, Anne E; Yde, Christina W

    2015-02-01

    Aromatase inhibitors (AIs) are used for treatment of estrogen receptor α (ER)-positive breast cancer; however, resistance is a major obstacle for optimal outcome. This preclinical study aimed at identifying potential new treatment targets in AI-resistant breast cancer cells. Parental MCF-7 breast cancer cells and four newly established cell lines, resistant to the AIs exemestane or letrozole, were used for a functional kinase inhibitor screen. A library comprising 195 different compounds was tested for preferential growth inhibition of AI-resistant cell lines. Selected targets were validated by analysis of cell growth, cell cycle phase distribution, protein expression, and subcellular localization. We identified 24 compounds, including several inhibitors of Aurora kinases e.g., JNJ-7706621 and barasertib. Protein expression of Aurora kinase A and B was found upregulated in AI-resistant cells compared with MCF-7, and knockdown studies showed that Aurora kinase A was essential for AI-resistant cell growth. In AI-resistant cell lines, the clinically relevant Aurora kinase inhibitors alisertib and danusertib blocked cell cycle progression at the G2/M phase, interfered with chromosome alignment and spindle pole formation, and resulted in preferential growth inhibition compared with parental MCF-7 cells. Even further growth inhibition was obtained when combining the Aurora kinase inhibitors with the antiestrogen fulvestrant. Our study is the first to demonstrate that Aurora kinase A and B may be treatment targets in AI-resistant cells, and our data suggest that therapy targeting both ER and Aurora kinases may be a potent treatment strategy for overcoming AI resistance in breast cancer.

  15. Investigating the Regulation and Potential Role of Nonhypoxic Hypoxia-Inducible Factor 1 (HIF-1) in Aromatase Inhibitor Resistant Breast Cancer

    DTIC Science & Technology

    2012-10-01

    respectively. Third , expression of HER2 and HIF-1 was also analyzed in another aromatase inhibitor (AI)-resistant cell line . Exemestane-resistant...combined with letrozole versus letrozole and placebo as first- line therapy for postmenopausal hormone receptor-positive metastatic breast cancer. J Clin...to identify other relevant factors involved that can be used as biomarkers of AI resistance or targets for therapy . One such factor may include HIF-1

  16. Aromatase inhibitors and bone loss.

    PubMed

    Perez, Edith A; Weilbaecher, Katherine

    2006-08-01

    The aromatase inhibitors (AIs) anastrozole (Arimidex), letrozole (Femara), and exemestane (Aromasin) are significantly more effective than the selective estrogen-receptor modulator (SERM) tamoxifen in preventing recurrence in estrogen receptor-positive early breast cancer. Aromatase inhibitors are likely to replace SERMs as first-line adjuvant therapy for many patients. However, AIs are associated with significantly more osteoporotic fractures and greater bone mineral loss. As antiresorptive agents, oral and intravenous bisphosphonates such as alendronate (Fosamax), risedronate (Actonel), ibandronate (Boniva), pamidronate (Aredia), and zoledronic acid (Zometa) have efficacy in preventing postmenopausal osteoporosis, cancer treatment-related bone loss, or skeletal complications of metastatic disease. Clinical practice guidelines recommend baseline and annual follow-up bone density monitoring for all patients initiating AI therapy. Bisphosphonate therapy should be prescribed for patients with osteoporosis (T score < -2.5) and considered on an individual basis for those with osteopenia (T score < -1). Modifiable lifestyle behaviors including adequate calcium and vitamin D intake, weight-bearing exercise, and smoking cessation should be addressed. Adverse events associated with bisphosphonates include gastrointestinal toxicity, renal toxicity, and osteonecrosis of the jaw. These safety concerns should be balanced with the potential of bisphosphonates to minimize or prevent the debilitating effects of AI-associated bone loss in patients with early, hormone receptor-positive breast cancer.

  17. New cell culture model for aromatase inhibitor-resistant breast cancer shows sensitivity to fulvestrant treatment and cross-resistance between letrozole and exemestane.

    PubMed

    Hole, Stine; Pedersen, Astrid M; Hansen, Susanne K; Lundqvist, Johan; Yde, Christina W; Lykkesfeldt, Anne E

    2015-04-01

    Aromatase inhibitor (AI) treatment is first-line systemic treatment for the majority of postmenopausal breast cancer patients with estrogen receptor (ER)-positive primary tumor. Although many patients benefit from treatment, some will develop resistance, and models mimicking acquired resistance will be valuable tools to unravel the resistance mechanisms and to find new treatments and biomarkers. Cell culture models for acquired resistance to the three clinically relevant AIs letrozole, anastrozole and exemestane were developed by selection and expansion of colonies of MCF-7 breast cancer cells surviving long-term AI treatment under conditions where endogenous aromatase-mediated conversion of androgen to estrogen was required for growth. Four cell lines resistant to each of the AIs were established and characterized. Maintenance of ER expression and function was a general finding, but ER loss was seen in one of twelve cell lines. HER receptor expression was increased, in particular EGFR expression in letrozole-resistant cell lines. The AI-resistant cell lines had acquired ability to grow without aromatase-mediated conversion of testosterone to estradiol, but upon withdrawal of AI treatment, testosterone induced minor growth stimulation. Letrozole, exemestane and tamoxifen were able to abrogate the testosterone stimulation but could not reduce growth to below the level in standard growth medium with AI, demonstrating cross-resistance between letrozole, exemestane and tamoxifen. In contrast, fulvestrant totally blocked growth of the AI resistant cell lines both after withdrawal of AI and with AI treatment. These data show that ER is the main driver of growth of the AI-resistant cell lines and indicate ligand-independent activation of ER. Fulvestrant is an efficient treatment option for these AI-resistant breast cancer cells, and the cell lines will be useful tools to disclose the underlying molecular mechanism for resistance to the different AIs.

  18. Aromatase inhibitors: possible future applications.

    PubMed

    Karaer, Oznur; Oruç, Semra; Koyuncu, Faik Mümtaz

    2004-08-01

    In premenopausal women ovaries are the major sites of estrogen production, while in postmenopausal women estrogen is produced by aromatization of ovarian and adrenal androgens in extragonadal sites, mostly in adipose tissue. Aromatase is a cytochrome P450 hemoprotein-containing enzyme complex that catalyzes the rate-limiting step in the conversion of androstenedione and testosterone to estrone and estradiol (E2). Aromatase inhibitors (AIs) have been developed primarily for use in either natural or surgical postmenopausal patients. In premenopausal women, the ovary can overcome the estrogen blockade by reflex increments of luteinizing hormone (LH) and follicle stimulating hormone (FSH), so AIs must be combined with a gonadotropin releasing hormone (GnRH) agonist to prevent the reflex LH and FSH increments. In advanced hormone-dependent breast cancer treatment, AIs have been shown to be superior to tamoxifen. Preliminary evidence also suggests superiority in the adjuvant, neoadjuvant settings and also for breast cancer prevention. AIs have been used in infertility and can increase ovulation rate. Reducing FSH dose, estrogen levels, improving response to FSH, implantation rates, and developing multiple follicles that can be used in in vitro maturation procedures are potential areas that AIs might be used in in assisted reproductive technologies (ART), besides simple ovulation induction. AIs are reported to be successful in treatment of endometriosis, an estrogen-dependent process. The use of AIs in gynecomastia, puberte precox, leiomyoma uteri, some estrogen-dependent cancers (ovarian), endometrial cancer and male infertility are reported; some of the results are promising but more clinical trials are needed. AIs are predicted to become the gold standard in the treatment of estrogen-dependent diseases in reproductive medicine in the near future.

  19. Aromatase inhibitors: structural features and biochemical characterization.

    PubMed

    Hong, Yanyan; Chen, Shiuan

    2006-11-01

    Aromatase is the enzyme synthesizing estrogens from androgens. In estrogen-dependent breast tumors, estrogens induce the expression of growth factors responsible for cancer cell proliferation. In situ estrogen synthesis by aromatase "is thought to play a key role in the promotion of breast cancer growth. Aromatase inhibitors (AIs) provide new approaches for the prevention and treatment of breast cancer by inhibiting estrogen biosynthesis. Through reverse transcription polymerase chain reaction (RT-PCR) and immunohistochemical techniques, aromatase has been found to be expressed in many endocrine tissues and tumors originating from these tissues. Unexpectedly, this enzyme is now known to also be expressed in liver, lung, and colon cancers. Such findings suggest a potential role for endocrine manipulation of these types of cancer using AIs. Three Food and Drug Administration (FDA)-approved AIs, anastrozole (Arimidex), letrozole (Femara), and exemestane (Aromasin), effectively challenging tamoxifen, have been used as first-line drugs in the treatment of hormone-dependent breast cancer, and possibly other aromatase-expressing cancers. In addition, natural anti-aromatase chemicals, such as flavones and coumarins, have been identified. Efforts to develop new lines of AIs derived from these phytochemicals have been initiated in several laboratories. Finally, significant progress has been made in the understanding of the structure-function relationship of aromatase. Such information has helped the examination of binding characteristics of AIs, the evaluation of reaction mechanism of aromatase, and the explanation of the molecular basis for a low catalytic activity of the natural variant, M364T.

  20. Anti-tumor efficacy of new 7α-substituted androstanes as aromatase inhibitors in hormone-sensitive and resistant breast cancer cells.

    PubMed

    Amaral, Cristina; Varela, Carla L; Maurício, João; Sobral, Ana Filipa; Costa, Saul C; Roleira, Fernanda M F; Tavares-da-Silva, Elisiário J; Correia-da-Silva, Georgina; Teixeira, Natércia

    2017-04-07

    The majority of breast cancer cases are estrogen receptor positive (ER(+)). Although, third-generation aromatase inhibitors (AIs) are used as first-line treatment in post-menopausal women, they cause endocrine resistance and bone loss, which limits their success. Therefore, there is a demand to discover new potent molecules, with less toxicity that can circumvent these drawbacks. Our group has previously demonstrated that new 7α-substituted steroidal molecules, 7α-(2ξ,3ξ-epoxypropyl)androsta-1,4-diene-3,17-dione (3), 7α-allylandrost-4-ene-3,17-dione (6), 7α-allylandrost-4-en-17-one (9), 7α-allyl-3-oxoandrosta-1,4-dien-17β-ol (10) and 7α-allylandrosta-1,4-diene-3,17-dione (12) are potent AIs in placental microsomes. In this work, it was investigated their anti-aromatase activity and in vitro effects in sensitive and resistant breast cancer cells. All the steroids efficiently inhibit aromatase in breast cancer cells, allowing to establish new structure-activity relationships for this class of compounds. Moreover, the new AIs can inhibit breast cancer cell growth, by causing cell cycle arrest and apoptosis. The effects of AIs 3 and 12 on sensitive cells were dependent on aromatase inhibition and androgen receptor (AR), while for AI 9 and AI 10 were AR- and ER-dependent, respectively. In addition, it was shown that all the AIs can sensitize resistant cancer cells being their behavior similar to the sensitive cells. In summary, this study contributes to the understanding of the structural modifications in steroidal scaffold that are translated into better aromatase inhibition and anti-tumor properties, providing important information for the rational design/synthesis of more effective AIs. In addition, allowed the discovery of new potent 7α-substituted androstane molecules to inhibit tumor growth and prevent endocrine resistance.

  1. Aromatase inhibitors: past, present and future.

    PubMed

    Séralini, G; Moslemi, S

    2001-06-10

    For the cellular physiology of sex steroid sensitive cells, the androgen/estrogen ratio may be more important than only one hormone action per se, in both sexes. This ratio is controlled in vertebrates by aromatase; its gene expression can be inhibited in different ways, and this is crucial for the treatment of estrogen-dependent diseases such as breast cancer, or gynecomastia in males for instance. To reach this goal, new steroidal and non-steroidal inhibitors are continuously being developed, and some of them are used as first or second line agents. Aromatase inhibition is also an essential tool for studying the role of estrogens in the adult, or during development. Aromatase inhibitors have shown in particular that estrogens are essential also in males for skeletal maturation and bone mineralization, development of masculine dendritic morphology in male brain linked to mating behaviour, and testicular function. Testosterone is often the prohormone converted in situ in active estrogens, at these levels. Several strategies can be used for aromatase inhibition. The first ones employed were blind screening or deductions from in vivo observations, which led for instance to the discovery of the role of aminoglutethimide in aromatase inhibition. Subsequently, in the years 1975-1990, the molecular modeling of compounds to mimic the substrate shape of the enzyme constituted the major idea. Hundreds of chemicals were synthesized by numerous authors, ranging from the well-known and very efficient 4-OHA to complicated imidazole or indane derivatives tested by sophisticated comparative molecular field analyses. Reticulum-bound active aromatase has not as yet been X-ray analyzed. Thus, aromatase inhibitors were also used more recently to probe and understand the active site conformation of the enzyme and its modelization was obtained from comparisons with bacterial-related cytochromes. We developed a mammalian model considerably closer to human aromatase in order to study the

  2. Binding characteristics of aromatase inhibitors and phytoestrogens to human aromatase.

    PubMed

    Chen, S; Kao, Y C; Laughton, C A

    1997-04-01

    We have evaluated the binding characteristics of three steroidal inhibitors [4-hydroxyandrostenedione (4-OHA), 7alpha-(4'-amino)phenylthio-1,4-androstadiene-3,17-dione (7alpha-APTADD), and bridge (2,19-methyleneoxy) androstene-3,17-dione (MDL 101,003)], four nonsteroidal inhibitors [aminoglutethimide (AG), CGS 20267, ICI D1033, and vorozole (R83842)], and two flavone phytoestrogens (chrysin, and 7,8-dihydroxyflavone) to aromatase through a combination of computer modeling and inhibitory profile studies on the wild-type and six aromatase mutants (I133Y, P308F, D309A, T310S, I395F, and I474Y). We have generated two aromatase models based on the x-ray structures of cytochrome P450-cam and cytochrome P450bm3, respectively. A major difference between the cytochrome P450cam-based and cytochrome P450bm3-based models is in the predicted lengths of helices F and G. In the cytochrome P450cam-based model, helices F and G lie antiparallel and extend across the active-site face of the molecule from one edge to the center, so that the carboxyl-terminal residues of helix F and the N-terminal residues of helix G make a major contribution to the structure of the active site. In the cytochrome P450bm3-based model, both helices are longer and so extend almost all the way across the active-site face of the molecule. Considering the size of the androgen substrate, we evaluated our results mainly based on the cytochrome P450cam model. The mutations involved in this study are thought to be at or near the proposed active site pocket. The inhibitory profile analysis has produced very interesting results and provided a molecular basis as to how seven aromatase inhibitors with different structures bind to the active site of aromatase. Furthermore, the investigation reveals that phytoestrogens bind to the active site of aromatase in a different orientation from that in the estrogen receptor.

  3. Androgen metabolite-dependent growth of hormone receptor-positive breast cancer as a possible aromatase inhibitor-resistance mechanism.

    PubMed

    Hanamura, Toru; Niwa, Toshifumi; Nishikawa, Sayo; Konno, Hiromi; Gohno, Tatsuyuki; Tazawa, Chika; Kobayashi, Yasuhito; Kurosumi, Masafumi; Takei, Hiroyuki; Yamaguchi, Yuri; Ito, Ken-Ichi; Hayashi, Shin-Ichi

    2013-06-01

    Aromatase inhibitors (AIs) have been reported to exert their antiproliferative effects in postmenopausal women with hormone receptor-positive breast cancer not only by reducing estrogen production but also by unmasking the inhibitory effects of androgens such as testosterone (TS) and dihydrotestosterone (DHT). However, the role of androgens in AI-resistance mechanisms is not sufficiently understood. 5α-Androstane-3β,17β-diol (3β-diol) generated from DHT by 3β-hydroxysteroid dehydrogenase type 1 (HSD3B1) shows androgenic and substantial estrogenic activities, representing a potential mechanism of AI resistance. Estrogen response element (ERE)-green fluorescent protein (GFP)-transfected MCF-7 breast cancer cells (E10 cells) were cultured for 3 months under steroid-depleted, TS-supplemented conditions. Among the surviving cells, two stable variants showing androgen metabolite-dependent ER activity were selected by monitoring GFP expression. We investigated the process of adaptation to androgen-abundant conditions and the role of androgens in AI-resistance mechanisms in these variant cell lines. The variant cell lines showed increased growth and induction of estrogen-responsive genes rather than androgen-responsive genes after stimulation with androgens or 3β-diol. Further analysis suggested that increased expression of HSD3B1 and reduced expression of androgen receptor (AR) promoted adaptation to androgen-abundant conditions, as indicated by the increased conversion of DHT into 3β-diol by HSD3B1 and AR signal reduction. Furthermore, in parental E10 cells, ectopic expression of HSD3B1 or inhibition of AR resulted in adaptation to androgen-abundant conditions. Coculture with stromal cells to mimic local estrogen production from androgens reduced cell sensitivity to AIs compared with parental E10 cells. These results suggest that increased expression of HSD3B1 and reduced expression of AR might reduce the sensitivity to AIs as demonstrated by enhanced androgen

  4. Presence of aromatase inhibitors in cycads.

    PubMed

    Kowalska, M T; Itzhak, Y; Puett, D

    1995-07-28

    Cycads, the most primitive of the living gymnosperms, have been used and continue to be used for food and medicinal purposes by many cultures, although toxins must be removed before ingestion. In our quest to identify tropical plants that contain inhibitors of the cytochrome P-450 aromatase and thus may be efficacious in treating estrogen-dependent tumors, we have screened extracts from 5 species of cycad folia encompassing 3 genera: Cycas cairnsiana F. Muell., Cycas revoluta Thunb., Cycas rumphii Miq., Dioon spinulosum Dyer and Encephalartos ferox Bertol. All extracts were found to contain inhibitors of the human enzyme.

  5. Aromatase inhibitors: assessment of biochemical efficacy measured by total body aromatase inhibition and tissue estrogen suppression.

    PubMed

    Lønning, Per E; Geisler, Jürgen

    2008-02-01

    The implementation of aromatase inhibitors for treatment of early and metastatic breast cancer has been one of the major improvements in endocrine therapy of breast cancer. Measurement of endocrine effects of aromatase inhibition in vivo has been a major tool in the process of evaluating novel compounds. Biochemical efficacy of aromatase inhibitors in vivo may be determined from their effects on "total body aromatization" as well changes in plasma and tissue estrogen levels. Due to high sensitivity, tracer methods allowing calculation of whole body aromatase inhibition are still considered the gold standard. The method developed by our group in collaboration with the Royal Marsden Hospital and the results of this joint program are summarized and discussed. These studies allowed classification of the different aromatase inhibitors and their optimal dosage, selecting the best compounds for clinical evaluation. In vivo total body aromatase assessment is a work-consuming method, allowing such studies to be conducted in a limited number of patients only. In contrast, plasma estrogen measurement is a cruder but simpler method, allowing screening of larger groups of patients. As plasma estrogens arise through passive diffusion of estrogens synthesized in different body compartments, plasma estrogens, as well as total body aromatase assessment, present a rough estimate of total body tissue estrogen production, and changes associated with treatment with aromatase inhibitors reflect the effects on tissue estrogen production in general. However, plasma estrogen levels do not correlate to breast cancer tissue estrogen levels. This is due to the endocrine autonomy of breast cancer tissue with significant local estrogen production in some tumors. Thus, direct measurement of intratumor estrogens is demanded to evaluate the effects of aromatase inhibitors in malignant target tissues. Our group has developed a highly sensitive HPLC-RIA for the simultaneous measurement of estrone

  6. Lead optimization of 4-imidazolylflavans: new promising aromatase inhibitors.

    PubMed

    Yahiaoui, Samir; Pouget, Christelle; Buxeraud, Jacques; Chulia, Albert José; Fagnère, Catherine

    2011-06-01

    Our previous studies have shown that several 7-substituted-4-imidazolylflavans are potent inhibitors of aromatase. These compounds were designed considering the anti-aromatase effect of some natural flavonoids and the importance of an azole ring for synthetic inhibitors such as letrozole or anastrozole towards binding to the heme iron of aromatase. In this study, we report the optimization of these lead compounds by the modulation of flavan A ring. The resulting 7,8-benzo-4-imidazolylflavans were tested in order to assess their ability to inhibit aromatase. Biological data concerning enantiomers obtained from the chiral separation of the racemate compound 4-imidazolyl-7-methoxyflavan are also presented.

  7. The Evolutionary Tale and Future Directions of Aromatase Inhibitors in Breast Carcinoma.

    PubMed

    Bhattacharjee, Dipanjan; Kumari, Meena K; Avin, S; Babu Amberkar, Mohan V

    2017-03-27

    Aromatase inhibitors have often been likened to that of 'medical scalpels' for the treatment of breast carcinoma. By inhibiting the singular step of aromatisation, they have proven to be extremely effective allies in the treatment of breast cancer among postmenopausal women. However, their relevance soon may not be limited to the post-menopausal age group alone. Recent studies have hinted at their utility amongst the pre-menopausal women; combined with ovarian ablation techniques, aromatase inhibitors may prove to be equally effective and more, as compared to tamoxifen in this age-group. Additionally, explorations aimed at ascertaining their potential utility as an effective preventive strategy against breast carcinoma have yielded encouraging results. However, for aromatase inhibitors to be able to attain their full potential, further strategic fine-tuning aimed at maximising their efficacy and minimising their potentially far-reaching adverse effects, is the need of the hour. Despite the recent diversification, the issue of resistance to aromatase inhibitors in breast cancer threatens to derail the advances so gained till date. Fortunately, a few novel ploys have come to the fore, for instance combining aromatase inhibitors with HER-2 antibodies that could potentially help circumvent the menace of resistance in the near future. Till date, the utility of aromatase inhibitors can at best be described as one-dimensional. However, with the unearthing of potential new avenues for its application, this assortment of molecules today stands on the precipice of ushering in a new revolution in the treatment of breast carcinom.

  8. Long-term complete remission of metastatic breast cancer, induced by a steroidal aromatase inhibitor after failure of a non-steroidal aromatase inhibitor

    PubMed Central

    Shioi, Yoshihiro; Kashiwaba, Masahiro; Inaba, Toru; Komatsu, Hideaki; Sugai, Tamotsu; Wakabayashi, Go

    2014-01-01

    Patient: Female, 56 Final Diagnosis: Breast cancer Symptoms: Solid mass in the right breast Medication: Exemestane Clinical Procedure: — Specialty: Oncology Objective: Unusual clinical course Background: The efficacy of third-generation aromatase inhibitors for hormone receptor-positive postmenopausal metastatic breast cancer is well established. Although several clinical trials have reported incomplete cross-resistance between different aromatase inhibitors, few cases of complete responses of recurrent metastatic breast cancer occurring after substituting a second aromatase inhibitor have been reported. We here present a rare case of non-steroidal aromatase inhibitor-tolerant metastatic breast cancer with long-term complete remission following substitution of a steroidal aromatase inhibitor. Case Report: We present the case of a 56-year-old Japanese woman who underwent right breast-conserving surgery for breast cancer, TNM staging T1, N0, M0, Stage I. She received adjuvant chemotherapy with 6 cycles of FEC100 and radiation therapy, and then began hormonal therapy with anastrozole. Twelve months postoperatively, computed tomography (CT) revealed multiple lung metastases. Exemestane was substituted for anastrozole. After 3 months of exemestane, CT showed that all lung metastases had completely resolved. Her complete response was maintained for 5 years: she died during a tsunami 6 years after the initial surgery. Conclusions: Substitution of a steroidal for a non-steroidal aromatase inhibitor produced a sustained complete remission in a patient with hormonal receptor-positive postmenopausal recurrent breast cancer. Achieving complete response after switching from a non-steroidal to a steroidal aromatase inhibitor in a hormonal receptor-positive postmenopausal recurrent breast cancer contributed to a higher quality of life for the patient. Further investigation is needed to identify the predictors of long-term remission following such a switch. PMID:24587856

  9. Bone scan alterations in aromatase inhibitor-treated patients.

    PubMed

    De Geeter, Frank; Van den Bruel, Annick; De Cuypere, Eveline; Langlois, Michel

    2015-01-01

    We report bone scan changes in 3 patients receiving aromatase inhibitors as adjuvant treatment for postmenopausal hormone receptor-positive breast cancer. Compared with bone scans before treatment, repeated scans after at least 10 months of aromatase inhibitor treatment showed increased activity in the peripheral skeleton and the skull. In 2 patients, these alterations could be correlated with increased markers of bone turnover. They probably result from high bone turnover induced by estrogen depletion caused by aromatase inhibitors. This effect should be taken into account in the differential diagnosis of a bone scan pattern suggestive of hyperparathyroidism, which was ruled out.

  10. Effectively nursing patients receiving aromatase inhibitor therapy.

    PubMed

    Wengström, Y

    2008-06-01

    Inhibiting estrogen production is a common means of preventing breast cancer recurrence. The aromatase inhibitors (AIs) are becoming the preferred treatment over tamoxifen as adjuvant therapy for postmenopausal women with hormone-sensitive early breast cancer. Like all adjuvant therapies, AIs have adverse events (AEs) associated with their use, many of which resemble symptoms common to menopause. Because of the greater efficacy of AIs in preventing breast cancer recurrence over tamoxifen, these AEs may be considered tolerable by many patients and often can be effectively managed and/or prevented. Educating patients about anticipated AEs may help them understand, accept, and cope with these AEs. This article reviews the AEs associated with different adjuvant AI treatments and highlights some strategies to manage them effectively. It also highlights the importance of patient education regarding AI therapy and involvement in treatment decisions, which may lead to better long-term adherence and ultimately to better outcomes.

  11. Aromatase inhibitors in stimulated IVF cycles

    PubMed Central

    2011-01-01

    Aromatase inhibitors have been introduced as a new treatment modality that could challenge clomiphene citrate as an ovulation induction regiment in patients with PCOS. Although several randomized trials have been conducted regarding their use as ovulation induction agents, only few trials are available regarding their efficacy in IVF stimulated cycles. Current available evidence support that letrozole may have a promising role in stimulated IVF cycles, either when administered during the follicular phase for ovarian stimulation. Especially for women with poor ovarian response, letrozole appears to have the potential to increase clinical pregnancy rates when combined with gonadotropins, whereas at the same time reduces the total gonadotropin dose required for ovarian stimulation. However, given that in all of the trials letrozole has been administered in GnRH antagonist cycles, it is intriguing to test in the future how it may perform when used in GnRH agonist cycles. Finally administration of letrozole during luteal phase in IVF cycles offers another treatment modality for patients at high risk for OHSS taking into account that it drastically reduces estradiol levels PMID:21693033

  12. Aromatase inhibitors from Urtica dioica roots.

    PubMed

    Gansser, D; Spiteller, G

    1995-04-01

    Methanolic extracts of stinging nettle (Urtica dioica L.) roots were investigated for aromatase inhibition. Enzyme inhibition was detected only after appropriate chromatographic separation. Inhibitory effects on aromatase could be demonstrated in vitro for a variety of compounds belonging to different classes. The following compounds developed weak to moderate activity: secoisolariciresinol, oleanolic and ursolic acid, (9Z,11E)-13-hydroxy-9,11-octadecadienoic acid, and 14-octacosanol (5). Inhibitory effects on aromatase have been known to date neither for pentacyclic triterpenes nor for secondary fatty alcohols. The potential physiological significance of the above findings is discussed. Compound 5 is a previously unknown constituent of plants.

  13. A yeast screen system for aromatase inhibitors and ligands for androgen receptor: yeast cells transformed with aromatase and androgen receptor.

    PubMed

    Mak, P; Cruz, F D; Chen, S

    1999-11-01

    Endocrine disruptors are hormone mimics that modify hormonal action in humans and animals. It is thought that some endocrine disruptors modify estrogen and androgen action in humans and animals by suppressing aromatase activity. Aromatase cytochrome P450 is the key enzyme that converts C19 androgens to aromatic C18 estrogenic steroids. We have developed a novel aromatase inhibitor screening method that allows us to identify antiaromatase activity of various environmental chemicals. The screen was developed by coexpressing the human aromatase and the mouse androgen receptor in yeast cells, which carry the androgen-responsive ss-galactosidase reporter plasmid. Functional expression of aromatase in yeast has been demonstrated using the [3H]-water release assay with intact cells as well as with yeast microsomes. The aromatase activity could be blocked by known aromatase inhibitors such as aminoglutethimide (AG). Yeast-produced androgen receptors were able to transactivate a yeast basal promoter linked to an androgen-responsive element in response to androgens. The resultant triple yeast transformant responded to the treatment of testosterone, androstenedione, or 5 alpha-dihydrotestosterone (5 alpha-DHT). In the absence of the aromatase inhibitor AG, transcriptional activation was observed only for the nonaromatizable androgen 5 alpha-DHT. However, the two aromatizable androgens (testosterone and androstenedione) induced the reporter activity in the presence of AG. Using this yeast-based assay, we confirmed that two flavones, chrysin and alpha-naphtholflavone, are inhibitors of aromatase. Thus, this yeast system allows us to develop a high-throughput screening method, without using radioactive substrate, to identify aromatase inhibitors as well as new ligands (nonaromatizable androgen mimics) for the androgen receptors. In addition, this screening method also allows us to distinguish nonandrogenic aromatase inhibitors from inhibitors with androgenic activity. This yeast

  14. The potency and clinical efficacy of aromatase inhibitors across the breast cancer continuum

    PubMed Central

    Lønning, P. E.

    2011-01-01

    The strategy of using estrogen suppression to treat breast cancer led to the development of aromatase inhibitors, including the third-generation nonsteroidal compounds anastrozole and letrozole, and the steroidal compound exemestane. Aromatase inhibitors potently inhibit aromatase activity and also suppress estrogen levels in plasma and tissue. In clinical studies in postmenopausal women with breast cancer, third-generation aromatase inhibitors were shown superior to tamoxifen for the treatment of metastatic disease. Studies of adjuvant therapy with aromatase inhibitors include (i) head-to-head studies of 5 years of the aromatase inhibitor versus 5 years of tamoxifen monotherapy; (ii) sequential therapy of 2–3 years of tamoxifen followed by an aromatase inhibitor (or the opposite sequence) versus 5 years of tamoxifen monotherapy; (iii) extended therapy with an aromatase inhibitor after 5 years of tamoxifen; and (iv) sequential therapy with an aromatase inhibitor versus aromatase inhibitor monotherapy. Recent results from the Arimidex, Tamoxifen, Alone or in Combination and Breast International Group 1–98 trials advocate using an aromatase inhibitor upfront. This article examines the clinical data with aromatase inhibitors, following a brief summary of their pharmacology. PMID:20616198

  15. Expression of the K303R Estrogen Receptor α Breast Cancer Mutation Induces Resistance to an Aromatase Inhibitor via Addiction to the PI3K/Akt Kinase Pathway

    PubMed Central

    Barone, Ines; Cui, Yukun; Herynk, Matthew H; Corona-Rodriguez, Arnoldo; Giordano, Cinzia; Selever, Jennifer; Beyer, Amanda; Andò, Sebastiano; Fuqua, Suzanne A. W.

    2009-01-01

    Aromatase inhibitors (AIs) are rapidly becoming the first choice for hormonal treatment of estrogen receptor alpha (ERα)-positive breast cancer in postmenopausal women. However, de novo and acquired resistance frequently occurs. We have previously identified a lysine to arginine transition at residue 303 (K303R) in ERα in premalignant breast lesions and invasive breast cancers, which confers estrogen hypersensitivity and resistance to tamoxifen treatment. Thus, we questioned whether resistance to AIs could arise in breast cancer cells expressing the ERα mutation. As preclinical models to directly test this possibility, we generated K303R-overexpressing MCF-7 cells stably transfected with an aromatase expression vector. Cells were stimulated with the aromatase substrate, androstenedione (AD), with or without the AI anastrozole (Ana). We found that Ana decreased AD-stimulated growth of WT cells, while K303R-expressing cells were resistant to the inhibitory effect of Ana on growth. We propose that a mechanism of resistance involves an increased binding between the mutant receptor and the p85α regulatory subunit of phosphatidylinositol-3-OH kinase (PI3K), leading to increased PI3K activity and activation of protein kinase B (PKB)/Akt survival pathways. Inhibition of the selective “addiction” to the PI3K/Akt pathway reversed AI resistance associated with expression of the mutant receptor. Our findings suggest that the K303R ERα mutation might be a new predictive marker of response to AIs in mutation-positive breast tumors, and that targeting the PI3K/Akt pathway may be a useful strategy for treating patients with tumors resistant to hormone therapy. PMID:19487288

  16. Aromatase Inhibitor Associated Musculoskeletal Symptoms are associated with Reduced Physical Activity among Breast Cancer Survivors

    PubMed Central

    Brown, Justin C.; Mao, Jun J.; Stricker, Carrie; Hwang, Wei-Ting; Tan, Kay-See; Schmitz, Kathryn H.

    2014-01-01

    Background Physical activity has numerous health benefits for breast cancer survivors. Recent data suggest that some breast cancer survivors treated with aromatase inhibitors may experience aromatase inhibitor associated musculoskeletal symptoms. It is unknown whether aromatase inhibitor associated musculoskeletal symptoms are associated with reduced physical activity and what other risk factors are associated with such physical activity reductions. Methods We conducted a cross-sectional study at a large university-based breast cancer clinic among breast cancer survivors prescribed an aromatase inhibitor. At routine follow-up, we surveyed participants about aromatase inhibitor associated musculoskeletal symptoms, as well as pre-aromatase inhibitor, and current, physical activity levels. Results Among 300 participants, 90 (30%) reported a reduction of physical activity since the initiation of aromatase inhibitor therapy. Those with aromatase inhibitor associated musculoskeletal symptoms were more likely to report decreased physical activity (62% versus 38%, p=0.001) compared to those without aromatase inhibitor associated musculoskeletal symptoms. In multivariate analyses, aromatase inhibitor associated musculoskeletal symptoms [odds ratio (OR) =2.29 (95% confidence interval (CI): 1.36–3.86)], and body mass index [OR=1.06 (95% CI: 1.02–1.12)] were associated with reductions in physical activity. In subgroup analysis among breast cancer survivors with aromatase inhibitor associated musculoskeletal symptoms, self-reported lower extremity joint pain [OR=1.23 (95% CI: 1.00–1.50)] and impaired lower extremity physical function [OR=1.07 (95% CI: 1.01–1.14)] were associated with reductions in physical activity. Conclusion Breast cancer survivors with aromatase inhibitor associated musculoskeletal symptoms were more likely to report reductions in physical activity since initiating aromatase inhibitor therapy compared to those without aromatase inhibitor associated

  17. Clinical utilities of aromatase inhibitors in breast cancer

    PubMed Central

    Chumsri, Saranya

    2015-01-01

    Aromatase is an enzyme that converts testosterones to estrogens. Inhibition of this enzyme has been shown to have several clinical utilities in breast cancer. Currently, there are three aromatase inhibitors (AIs) in clinical use, namely anastrozole, letrozole, and exemestane. AIs have been used in various clinical settings for breast cancer, ranging from chemoprevention in breast cancer to treating breast cancer in both early stage in the adjuvant setting and metastatic disease. This article reviews mechanism of action, AI classification, and clinical utilities of AIs in various clinical settings in the context of breast cancer. PMID:26005359

  18. Molecular modeling evaluation of non-steroidal aromatase inhibitors.

    PubMed

    Narayana, Bheemanapalli Lakshmi; Pran Kishore, Deb; Balakumar, Chadrasekaran; Rao, Kaki Venkata; Kaur, Rajwinder; Rao, Akkinepally Raghuram; Murthy, Javali Narashima; Ravikumar, Muttineni

    2012-05-01

    A recent discovery of aromatase crystal structure triggered the efforts to design novel aromatase inhibitors for breast cancer therapy. While correlating docking scores with inhibitory potencies of known ligands, feeble robustness of scoring functions toward prediction was observed. This prompted us to develop new prediction models using stepwise regression analysis based on consensus of different docking and their scoring methods (GOLD, LIGANDFIT, and GLIDE). Quantitative structure-activity relationships were developed between the aromatase inhibitory activity (pIC(50) ) of flavonoid derivatives (n=39) and docking scores and docking descriptors. QSAR models have been validated internally [using leave-one-out cross-validated r(2)(cv) (LOO-Q2))] and externally to ensure the predictive capacity of the models. Model 2 [M2] developed using consensus of docking scores of scoring functions viz. ASP, potential of mean force and DOCK Score (r(2)(cv)=0.850, r(2) = 0.870, r(2)(pred) = 0.633, RMSE = 0.363 μm, r(2)(m(test)) =0.831, r(2)(m(overall)) =0.832) was found to be better in predicting aromatase inhibitory potency (pIC(50) ) compared to the Model 1 [M1] based on docking descriptors (r(2)(cv)= 0.848, r(2) = 0.825, r(2)(pred) =0.788, RMSE=0.421μm, r(2)(m(test)) =0.808, r(2)(m(overall)) =0.821). It has been observed that the natural flavonoids and their derivatives were less potent compared to these scaffolds with imidazolylmethyl substitution owing to the interaction of nitrogen atom of the imidazole ring toward the heme (Fe(3+) ) of the aromatase. Results confirm the potential of our methodology for the design of new potent non-steroidal aromatase inhibitors.

  19. Nonsteroidal aromatase inhibitors for the treatment of breast cancer: an update.

    PubMed

    Gobbi, Silvia; Rampa, Angela; Belluti, Federica; Bisi, Alessandra

    2014-01-01

    Estrogens are known to be important in breast cancer growth in both pre- and post-menopausal women. Although circulating estrogen concentrations are very low after menopause, peripheral tissues generate sufficient concentrations to stimulate tumor growth. As aromatase is the rate-limiting enzyme in estrogen biosynthesis, inhibitors of this enzyme represent effective targeted therapy for breast cancer. Three compounds are now FDA approved and have become the first-choice endocrine drugs for postmenopausal breast cancer patients, since they are associated with superior activity and better general tolerability when compared with the estrogen receptor modulator tamoxifen. Nevertheless, some questions concerning the use of aromatase inhibitors for the treatment of breast cancer still need to be addressed, mainly related to their side-effects and the development of resistance, making research in this field still appealing. Many research groups, including our own, are still dealing with the search of new compounds that possess aromatase inhibitory properties. In this review an update of the latest achievements in the field of nonsteroidal aromatase inhibitors will be given.

  20. New 7,8-benzoflavanones as potent aromatase inhibitors: synthesis and biological evaluation.

    PubMed

    Yahiaoui, Samir; Fagnere, Catherine; Pouget, Christelle; Buxeraud, Jacques; Chulia, Albert-José

    2008-02-01

    Some natural compounds such as flavonoids are known to possess a moderate inhibitory activity against aromatase, this enzyme being an interesting target for hormone-dependent breast cancer treatment. It has been demonstrated that the modulation of flavonoid skeleton could increase anti-aromatase effect. Therefore, new 7,8-benzoflavanones were synthesized and tested for their activity toward aromatase inhibition. It was observed that the introduction of a benzo ring at position C-7 and C-8 on flavanone skeleton led to new potent aromatase inhibitors, the resulting 7,8-benzoflavanones being until nine times more potent than aminogluthetimide (the first aromatase inhibitor used clinically).

  1. A yeast screen system for aromatase inhibitors and ligands for androgen receptor: yeast cells transformed with aromatase and androgen receptor.

    PubMed Central

    Mak, P; Cruz, F D; Chen, S

    1999-01-01

    Endocrine disruptors are hormone mimics that modify hormonal action in humans and animals. It is thought that some endocrine disruptors modify estrogen and androgen action in humans and animals by suppressing aromatase activity. Aromatase cytochrome P450 is the key enzyme that converts C19 androgens to aromatic C18 estrogenic steroids. We have developed a novel aromatase inhibitor screening method that allows us to identify antiaromatase activity of various environmental chemicals. The screen was developed by coexpressing the human aromatase and the mouse androgen receptor in yeast cells, which carry the androgen-responsive ss-galactosidase reporter plasmid. Functional expression of aromatase in yeast has been demonstrated using the [3H]-water release assay with intact cells as well as with yeast microsomes. The aromatase activity could be blocked by known aromatase inhibitors such as aminoglutethimide (AG). Yeast-produced androgen receptors were able to transactivate a yeast basal promoter linked to an androgen-responsive element in response to androgens. The resultant triple yeast transformant responded to the treatment of testosterone, androstenedione, or 5 alpha-dihydrotestosterone (5 alpha-DHT). In the absence of the aromatase inhibitor AG, transcriptional activation was observed only for the nonaromatizable androgen 5 alpha-DHT. However, the two aromatizable androgens (testosterone and androstenedione) induced the reporter activity in the presence of AG. Using this yeast-based assay, we confirmed that two flavones, chrysin and alpha-naphtholflavone, are inhibitors of aromatase. Thus, this yeast system allows us to develop a high-throughput screening method, without using radioactive substrate, to identify aromatase inhibitors as well as new ligands (nonaromatizable androgen mimics) for the androgen receptors. In addition, this screening method also allows us to distinguish nonandrogenic aromatase inhibitors from inhibitors with androgenic activity. This yeast

  2. Molecular basis for the interaction of four different classes of substrates and inhibitors with human aromatase.

    PubMed

    Hong, Yanyan; Cho, Michael; Yuan, Yate-Ching; Chen, Shiuan

    2008-03-01

    Aromatase cytochrome P450 (CYP19) converts androgen to estrogen. In this study, the interactions of four classes of compounds, 17beta-estradiol (the product of aromatase), 17-methyltestosterone (a synthetic androgen), dibenzylfluorescein (a synthetic substrate of aromatase), and coumestrol (a phytoestrogen), with aromatase were investigated through spectral analysis using purified human recombinant aromatase and site-directed mutagenesis studies using CHO cells expressing wild-type human aromatase or five aromatase mutants, E302D, D309A, T310S, S478T and H480Q. Spectral analysis showed that a type I binding spectrum was produced by the binding of 17-methyltestosterone to aromatase and a novel binding spectrum of aromatase was induced by dibenzylfluorescein. Mutagenesis experiments demonstrated that residues S478 and H480 in the beta-4 sheet play an important role in the binding of all four compounds. Computer-assisted docking of these compounds into the three-dimensional model of aromatase revealed that: (1) weak interaction between 17beta-estradiol and the beta-4 sheet of aromatase facilitates the release of 17beta-estradiol from the active site of aromatase; (2) 17-methyl group of 17-methyltestosterone affects its binding to aromatase; (3) dibenzylfluorescein binds to the active site of aromatase with its O-dealkylation site near the heme iron and residue T310; and (4) coumestrol binds to aromatase in a manner such that rings A and C of coumestrol mimic rings A and B of steroid. These structure-function studies help us to evaluate the structural model of aromatase, and to accelerate the structure-based design for new aromatase inhibitors.

  3. [Progress in study of the structure, catalytic mechanism and inhibitors of aromatase].

    PubMed

    Fu, Jing; Shen, Zhong-Hua; Cheng, Fei-Xiong; Liu, Gui-Xia; Li, Wei-Hua; Tang, Yun

    2012-01-01

    Aromatase is a key enzyme responsible for in vivo estrogen biosynthesis. Inhibition of the activity of the aromatase has become an alterative way for treatment of breast cancer. In this review, the structure and catalytic mechanism of the aromatase is briefly introduced followed by thorough review of the progress in the study of the steroidal and non-steroidal aromatase inhibitors. This review is focused on the natural compounds that exhibit the aromatase inhibition, which include flavonoids, xanthones, coumarins, and sesquiterpenes. The structure-activity relationship of these compounds is also discussed.

  4. Aromatase inhibitors and their future role in post-menopausal women with early breast cancer.

    PubMed Central

    Lønning, P. E.

    1998-01-01

    Anastrozole is the first aromatase inhibitor to show a significant survival advantage over megestrol acetate in post-menopausal women with advanced breast cancer. The rationale for extending the use of aromatase inhibitors to the treatment of early breast cancer is based on the efficacy observed in the advanced setting, combined with good tolerability and a convenient dosing regimen. Furthermore, oestrogen deprivation by ovarian ablation (similar to oestrogen antagonism with tamoxifen) is already established as an effective adjuvant treatment in premenopausal women with modality breast cancer. Anastrozole produces a profound suppression of plasma oestrogen levels which is greater than that obtained with earlier aromatase inhibitors (formestane, aminoglutethimide) or megestrol acetate. This could account for the differences in clinical efficacy seen between anastrozole and megestrol acetate. In terms of benefits over other endocrine agents, anastrozole causes significantly less weight gain than megestrol acetate; it does not have the partial agonist activity of tamoxifen, and is unlikely to lead to tumour stimulation in patients resistant to tamoxifen or to exert proliferative effects on the endometrium. The lack of oestrogen agonist activity, however, may possibly have detrimental effects on bone mineral density and blood lipid profile. Current clinical trials are investigating the efficacy and safety of anastrozole in the early breast cancer setting. The results of these trials will help to determine whether anastrozole has any benefits over tamoxifen, the current treatment of choice in post-menopausal women with early breast cancer. PMID:9741783

  5. TRPA1 Mediates Aromatase Inhibitor-Evoked Pain by the Aromatase Substrate Androstenedione.

    PubMed

    De Logu, Francesco; Tonello, Raquel; Materazzi, Serena; Nassini, Romina; Fusi, Camilla; Coppi, Elisabetta; Li Puma, Simone; Marone, Ilaria M; Sadofsky, Laura R; Morice, Alyn H; Susini, Tommaso; Terreni, Alessandro; Moneti, Gloriano; Di Tommaso, Mariarosaria; Geppetti, Pierangelo; Benemei, Silvia

    2016-12-01

    Aromatase inhibitors (AI) induce painful musculoskeletal symptoms (AIMSS), which are dependent upon the pain transducing receptor TRPA1. However, as the AI concentrations required to engage TRPA1 in mice are higher than those found in the plasma of patients, we hypothesized that additional factors may cooperate to induce AIMSS. Here we report that the aromatase substrate androstenedione, unique among several steroid hormones, targeted TRPA1 in peptidergic primary sensory neurons in rodent and human cells expressing the native or recombinant channel. Androstenedione dramatically lowered the concentration of letrozole required to engage TRPA1. Notably, addition of a minimal dose of androstenedione to physiologically ineffective doses of letrozole and oxidative stress byproducts produces AIMSS-like behaviors and neurogenic inflammatory responses in mice. Elevated androstenedione levels cooperated with low letrozole concentrations and inflammatory mediators were sufficient to provoke AIMSS-like behaviors. The generation of such painful conditions by small quantities of simultaneously administered TRPA1 agonists justifies previous failure to identify a precise link between AIs and AIMSS, underscoring the potential of channel antagonists to treat AIMSS. Cancer Res; 76(23); 7024-35. ©2016 AACR.

  6. Recent developments in steroidal and nonsteroidal aromatase inhibitors for the chemoprevention of estrogen-dependent breast cancer.

    PubMed

    Ahmad, Irshad; Shagufta

    2015-09-18

    Aromatase, a cytochrome P450 enzyme complex present in breast tissues, plays a significant role in the biosynthesis of important endogenous estrogens from androgens. The source of estrogen production in breast cancer tissues is intra-tumoral aromatase, and inhibition of aromatase may inhibit the growth stimulation effect of estrogens in breast cancer tissues. Consequently, aromatase is considered a useful therapeutic target in the treatment and prevention of estrogen-dependent breast cancer. Recently, different natural products and synthetic compounds have been rapidly developed, studied, and evaluated for aromatase inhibitory activity. Aromatase inhibitors are classified into two categories on the basis of their chemical structures, i.e., steroidal and nonsteroidal aromatase inhibitors. This review highlights the synthetic steroidal and nonsteroidal aromatase inhibitors reported in the literature in the last few years and will aid medicinal chemists in the design and synthesis of novel and pharmacologically-potent aromatase inhibitors for the treatment of breast cancer.

  7. Developing steroidal aromatase inhibitors-an effective armament to win the battle against breast cancer.

    PubMed

    Yadav, Mange Ram; Barmade, Mahesh A; Tamboli, Riyaj S; Murumkar, Prashant R

    2015-11-13

    Breast cancer, an emerging disease among the women population, occurs due to overexpression of estrogens. The enzyme aromatase plays a key rate limiting role in the biosynthesis of estrogens. Certain clinical advantages of the use of exemestane, a steroidal aromatase inhibitor over non-steroidal aromatase inhibitors have drawn the attention of researchers for the development of novel steroidal aromatase inhibitors.The current review is a humble attempt to compile the reports by various researchers till date on the synthesis of steroidal aromatase inhibitors. It has been tried to encompass the structural modifications carried out by various researchers in the steroid ring system by taking up the functional group modifications on rings A, B, ring A/B junction, ring-D, ring modifications, bridged derivatives and heterocyclic ring-fused derivatives in a systematic way.

  8. Nelumal A, the active principle of Ligularia nelumbifolia, is a novel aromatase inhibitor.

    PubMed

    Epifano, Francesco; Genovese, Salvatore; Fiorito, Serena; Nde, Chantal Magne; Clyne, Colin

    2014-06-01

    Nelumal A, the active principle of Ligularia nelumbifolia was preliminarily tested as an aromatase inhibitors in HEK293 cells transfected with aromatase cDNA and using anastrazole as the reference drug. This screening revealed that it showed an appreciable level of inhibition. Subsequent experiments aimed to evaluate the aromatase activity and expression in KGN cells confirmed that the title natural product, after an incubation of 48 h, compared favourably with anastrazole (1 microM) in the concentration range 10-30 microM. Moreover, nelumal A (30 microM) abolished the aromatase mRNA expression in the same cell line.

  9. Mechanism-based Categorization of Aromatase Inhibitors: A Potential Discovery and Screening Tool

    EPA Science Inventory

    Cytochrome P450 aromatase is a key steroidogenic enzyme that converts androgens to estrogens in vertebrates. There is much interest in aromatase inhibitors (AIs) because a number of environmental contaminants can act as AIs, thereby disrupting endocrine function in humans and wil...

  10. Successful use of aromatase inhibitor letrozole in NOA with an elevated FSH level: a case report.

    PubMed

    Zhao, D; Pan, L; Zhang, F; Pan, F; Ma, J; Zhang, X; Liu, Y

    2014-05-01

    Aromatase inhibitors inhibit the conversion of testosterone to oestrogens and could reduce serum oestradiol concentrations. Letrozole is one of aromatase inhibitors frequently used in treatment of men with oligospermia. We present the case of an infertile man with small testes and an elevated FSH level, which was diagnosed as NOA, hypospermatogenesis proven by testicular biopsy. After taking letrozole for 3 months, semen analyses by computer-aided sperm analysis present that this man had normal spermatogenesis. This is the first case report of the activation of spermatogenesis, in man who was NOA with elevated FSH level, resulting from the use of the one of aromatase inhibitors.

  11. Fetal safety profile of aromatase inhibitors: Animal data.

    PubMed

    Tiboni, Gian Mario; Ponzano, Adalisa

    2016-12-01

    Aromatase inhibitors (AIs) are a class of drugs that act by blocking the production of estrogens from androgens. The current review concentrates on the prenatal developmental toxicity of AIs in experimental models. Available data indicate that AIs may affect pregnancy at human therapeutic or lower doses. The window of vulnerability to AIs is not limited to organogenesis, but also includes the preimplantation stage and fetal periods. Decreased embryo/fetal survival was the prominent treatment-related effect. Morphological anomalies noted in fetuses exposed to AIs included skeletal anomalies, abnormal head morphology, increased ano-genital distance in female fetuses, and minor urinary tract system anomalies. Placental enlargement was consistently reported in rats and non-human primates after maternal treatment with several AIs. In conclusion, data from basic scientific research suggest that low intensity exposure to AIs applied during a wide gestational window can profoundly affect prenatal development.

  12. Design, synthesis, and 3D QSAR of novel potent and selective aromatase inhibitors.

    PubMed

    Leonetti, Francesco; Favia, Angelo; Rao, Angela; Aliano, Rosaria; Paluszcak, Anja; Hartmann, Rolf W; Carotti, Angelo

    2004-12-30

    The design, synthesis, and biological evaluation of a series of new aromatase inhibitors bearing an imidazole or triazole ring linked to a fluorene (A), indenodiazine (B), or coumarin scaffold (C) are reported. Properly substituted coumarin derivatives displayed the highest aromatase inhibitory potency and selectivity over 17-alpha-hydroxylase/17-20 lyase. The modeling of the aromatase inhibition data by Comparative Molecular Field Analysis (CoMFA/GOLPE 3D QSAR approach) led to the development of a PLS model with good fitting and predictive powers (n = 22, ONC = 3, r(2) = 0.949, s = 0.216, and q(2) = 0.715). The relationship between aromatase inhibition and the steric and electrostatic fields generated by the examined azole inhibitors enables a clear understanding of the nature and spatial location of the main interactions modulating the aromatase inhibitory potency.

  13. Facile synthesis of chrysin-derivatives with promising activities as aromatase inhibitors.

    PubMed

    Mohammed, Hamdoon A; Ba, Lalla A; Burkholz, Torsten; Schumann, Elena; Diesel, Britta; Zapp, Josef; Kiemer, Alexandra K; Ries, Christina; Hartmann, Rolf W; Hosny, Mohammed; Jacob, Claus

    2011-01-01

    Flavones such as chrysin show structural similarities to androgens, the substrates of human aromatase, which converts androgens to estrogens. Aromatase is a key target in the treatment of hormone-dependent tumors, including breast cancer. Flavone-based aromatase inhibitors are of growing interest, and chrysin in particular provides a (natural) lead structure. This paper reports multicomponent synthesis as a means for facile modification of the chrysin core structure in order to add functional elements. A Mannich-type reaction was used to synthesize a range of mono- and disubstituted chrysin derivatives, some of which are more effective aromatase inhibitors than the benchmark compound, aminoglutethimide. Similarly, the reaction of chrysin with various isonitriles and acetylene dicarboxylates results in a new class of flavone derivatives, tricyclic pyrano-flavones which also inhibit human aromatase. Multicomponent reactions involving flavones therefore enable the synthesis of a variety of derivatives, some of which may be useful as anticancer agents.

  14. Use of Network Inference to Unravel the Mechanisms of Action and Specificity of Aromatase Inhibitors

    EPA Science Inventory

    The vertebrate hypothalamus-pituitary-gonadal (HPG) axis is controlled through various feedback mechanisms in order to maintain a dynamic homeostasis during changing environmental conditions, including exposure to chemical stressors. In this study, three aromatase inhibitors, fad...

  15. Bilateral de quervain syndrome after aromatase inhibitor administration: a case report and review of the literature.

    PubMed

    Papadimitriou, Konstantinos; Kountourakis, Panteleimon; Morakis, Emmanouil; Vassiliou, Vassilios; Barbounis, Vasileios; Ardavanis, Alexandros

    2012-01-01

    Aromatase inhibitors are widely used as one of the main treatment options of both early and advanced hormone receptor-positive breast cancer in postmenopausal women. Unfortunately, musculoskeletal symptoms are often presented in patients treated with aromatase inhibitors (AIs), and, although the pathogenesis is unknown, postulated mechanisms have been described. Herein, to our knowledge, we present the first report of bilateral De Quervain syndrome related with AIs therapy with a review of the relevant literature.

  16. Bilateral De Quervain Syndrome after Aromatase Inhibitor Administration: A Case Report and Review of the Literature

    PubMed Central

    Papadimitriou, Konstantinos; Kountourakis, Panteleimon; Morakis, Emmanouil; Vassiliou, Vassilios; Barbounis, Vasileios; Ardavanis, Alexandros

    2012-01-01

    Aromatase inhibitors are widely used as one of the main treatment options of both early and advanced hormone receptor-positive breast cancer in postmenopausal women. Unfortunately, musculoskeletal symptoms are often presented in patients treated with aromatase inhibitors (AIs), and, although the pathogenesis is unknown, postulated mechanisms have been described. Herein, to our knowledge, we present the first report of bilateral De Quervain syndrome related with AIs therapy with a review of the relevant literature. PMID:22567020

  17. Different catalytic properties and inhibitor responses of the goldfish brain and ovary aromatase isozymes.

    PubMed

    Zhao, J; Mak, P; Tchoudakova, A; Callard, G; Chen, S

    2001-08-01

    The brain and ovarian aromatase isozymes of goldfish (Carassius auratus) are encoded by different CYP19 genes. This study measured aromatase activity in the goldfish brain tissues. For a direct comparison of the properties of the two aromatase isozymes, Chinese hamster ovary cells were stably transfected with brain- and ovary-derived cDNAs (respectively, p450 arom B and -A) and the properties of the expressed isozymes were compared. The kinetic parameters of the two isozymes were determined using androstenedione and testosterone as substrates and compared to those of human aromatase. Inhibition profile analyses on the two isozymes were performed using seven inhibitors [4-hydroxyandrostenedione, 7 alpha-(4'-amino)phenylthio-1,4-androstadiene-3,17-dione, bridge (2,19-methyleneoxy)androstene-3,17-dione, aminoglutethimide (AG), CGS 20267, ICI D1033, and vorozole]. Except for AG, the compounds tested were found to be much stronger inhibitors against the ovary enzyme than the brain enzyme. In addition, the ovary isoform was more sensitive to two phytoestrogens, chrysin and 7,8-dihydroxyflavone, than the brain form. These studies reveal that catalytic properties of the goldfish aromatase isoforms are significantly different from those of human aromatase. In addition, differences in the K(i) values of aromatase inhibitors for the two goldfish isoforms suggest structural variance in the active sites of these isozymes.

  18. Targeting the receptor tyrosine kinase RET in combination with aromatase inhibitors in ER positive breast cancer xenografts

    PubMed Central

    Fearns, Antony; Martin, Lesley-Ann; Chiarugi, Paola; Isacke, Clare M.; Morandi, Andrea

    2016-01-01

    The majority of breast cancers are estrogen receptor positive (ER+). Blockade of estrogen biosynthesis by aromatase inhibitors (AIs) is the first-line endocrine therapy for post-menopausal women with ER+ breast cancers. However, AI resistance remains a major challenge. We have demonstrated previously that increased GDNF/RET signaling in ER+ breast cancers promotes AI resistance. Here we investigated the efficacy of different small molecule RET kinase inhibitors, sunitinib, cabozantinib, NVP-BBT594 and NVP-AST487, and the potential of combining a RET inhibitor with the AI letrozole in ER+ breast cancers. The most effective inhibitor identified, NVP-AST487, suppressed GDNF-stimulated RET downstream signaling and 3D tumor spheroid growth. Ovariectomized mice were inoculated with ER+ aromatase-overexpressing MCF7-AROM1 cells and treated with letrozole, NVP-AST487 or the two drugs in combination. Surprisingly, the three treatment regimens showed similar efficacy in impairing MCF7-AROM1 tumor growth in vivo. However in vitro, NVP-AST487 was superior to letrozole in inhibiting the GDNF-induced motility and tumor spheroid growth of MCF7-AROM1 cells and required in combination with letrozole to inhibit GDNF-induced motility in BT474-AROM3 aromatase expressing cells. These data indicate that inhibiting RET is as effective as the current therapeutic regimen of AI therapy but that a combination treatment may delay cancer cell dissemination and metastasis. PMID:27602955

  19. Aromatase inhibitor-induced joint pain: melatonin's role.

    PubMed

    Burk, R

    2008-12-01

    Aromatase inhibitors (AIs) enjoy increasing use in breast cancer adjuvant therapy. But the joint pain associated with AIs significantly reduces patient adherence despite the clear survival benefits of this class of drugs. Two clues point to a novel hypothesis for this unexplained symptom. First, realizing that joint pain is associated with virtually all estrogen-depleting breast cancer treatments suggests that the cause is broader than this particular class of drugs. Second, the strongly circadian nature of these symptoms suggests circadian hormone involvement. This puts new light on some existing research findings: that estrogen depletion can increase pineal melatonin, that the ability of light to suppress pineal melatonin is more variable than once thought, and that an altered melatonin cycle is associated with rheumatoid arthritis patients, where identical circadian symptoms present. It is hypothesized that when AIs decrease estrogen levels, light-induced melatonin suppression (LIMS) loses efficacy, leading to an abnormal melatonin cycle as seen in rheumatoid arthritis patients, producing (via mechanisms not yet understood) the symptoms of morning stiffness. Not all frequencies of retinal light are equally effective at suppressing pineal melatonin; most artificial lighting has less relevant spectral density than sunlight. This hypothesis predicts that some patients can suppress the circadian joint pain associated with aromatase inhibitors merely by getting sufficient hours of daily retinal sunlight. A single patient history is discussed, in which a series of treatments had no effect on AI joint pain, while extended exposure to sunlight produced a definitive elimination of symptoms the next morning. To conclusively demonstrate the role of melatonin, light-emitting diodes of an appropriate frequency were mounted on a cap for the patient to wear. If worn first thing in the morning, the cap sharply curtailed the duration of morning stiffness. If worn for a

  20. 3D QSAR studies, pharmacophore modeling and virtual screening on a series of steroidal aromatase inhibitors.

    PubMed

    Xie, Huiding; Qiu, Kaixiong; Xie, Xiaoguang

    2014-11-14

    Aromatase inhibitors are the most important targets in treatment of estrogen-dependent cancers. In order to search for potent steroidal aromatase inhibitors (SAIs) with lower side effects and overcome cellular resistance, comparative molecular field analysis (CoMFA) and comparative molecular similarity indices analysis (CoMSIA) were performed on a series of SAIs to build 3D QSAR models. The reliable and predictive CoMFA and CoMSIA models were obtained with statistical results (CoMFA: q² = 0.636, r²(ncv) = 0.988, r²(pred) = 0.658; CoMSIA: q² = 0.843, r²(ncv) = 0.989, r²(pred) = 0.601). This 3D QSAR approach provides significant insights that can be used to develop novel and potent SAIs. In addition, Genetic algorithm with linear assignment of hypermolecular alignment of database (GALAHAD) was used to derive 3D pharmacophore models. The selected pharmacophore model contains two acceptor atoms and four hydrophobic centers, which was used as a 3D query for virtual screening against NCI2000 database. Six hit compounds were obtained and their biological activities were further predicted by the CoMFA and CoMSIA models, which are expected to design potent and novel SAIs.

  1. Steroidal pyrazolines evaluated as aromatase and quinone reductase-2 inhibitors for chemoprevention of cancer.

    PubMed

    Abdalla, Mohamed M; Al-Omar, Mohamed A; Bhat, Mashooq A; Amr, Abdel-Galil E; Al-Mohizea, Abdullah M

    2012-05-01

    The aromatase and quinone reductase-2 inhibition of synthesized heterocyclic pyrazole derivatives fused with steroidal structure for chemoprevention of cancer is reported herein. All compounds were interestingly less toxic than the reference drug (Cyproterone(®)). The aromatase inhibitory activities of these compounds were much more potent than the lead compound resveratrol, which has an IC(50) of 80 μM. In addition, all the compounds displayed potent quinone reductase-2 inhibition. Initially the acute toxicity of the compounds was assayed via the determination of their LD(50). The aromatase and quinone reductase-2 inhibitors resulting from this study have potential value in the treatment and prevention of cancer.

  2. Management of arthralgias associated with aromatase inhibitor therapy.

    PubMed

    Thorne, C

    2007-12-01

    For the upfront adjuvant therapy of postmenopausal estrogen receptor-positive breast cancer, the third-generation aromatase inhibitors (AIS) have shown a more favourable overall risk-benefit profile than has tamoxifen. Benefits of the ais include less frequent gynecologic, cerebrovascular, and thromboembolic adverse events; greater disease-free survival; and lower tumour recurrence. Although approximately 25% of postmenopausal women with early breast cancer report experiencing symptoms of arthralgia with ai therapy, 68-month data from the Arimidex, Tamoxifen, Alone or in Combination trial showed that, compared with tamoxifen, anastrozole treatment was associated with only a modest increase in the incidence of joint symptoms. The events, which were mostly mild-to-moderate in intensity, led to treatment withdrawal in 2% of patients on anastrozole as compared with 1% in the tamoxifen arm. The symptoms and changes correlate with clinical, biochemical, and radiologic findings in symptomatic women. To determine appropriate intervention, it is therefore essential to perform a comprehensive evaluation of musculoskeletal complaints to distinguish natural menopause-related degenerative disease from AI-related effects. The present review explores the advantages of differential diagnosis with an emphasis on history and physical and musculoskeletal examination; laboratory investigations are used to corroborate or rule out clinical impressions. The transient symptoms associated with the ais are manageable with an appropriate combination of lifestyle changes, including exercise and joint protection in conjunction with pharmacologic approaches.

  3. The role of aromatase inhibitors in early breast cancer.

    PubMed

    Chung, Cathie T; Carlson, Robert W

    2003-04-01

    The role of hormonal therapy for the treatment of patients with early stage breast cancer has been evaluated in many studies. The results of these studies establish tamoxifen as the gold standard of hormonal therapy for the adjuvant treatment of hormone receptor-positive invasive breast cancer in pre- and postmenopausal women. Studies show tamoxifen reduces the risk of invasive breast cancer in women at increased risk for the disease, including women with ductal carcinoma in situ. Tamoxifen has adverse effects such as hot flashes, increased risk of uterine cancer in postmenopausal women, and rare occurrence of thromboembolic disease. Despite the multiple therapeutic roles of tamoxifen, alternatives are needed. Aromatase inhibitors (AI) are drugs with antiestrogenic activity. AIs function by inhibiting the peripheral conversion of adrenally synthesized androstenedione to estradiol through inhibition of the aromatase enzyme. AIs do not suppress estradiol synthesis by the ovary adequately. Therefore, AIs are effective in reducing circulating estradiol levels in postmenopausal women, but not premenopausal women. Selective nonsteroidal AIs, including anastrozole (Arimidex; AstraZeneca, Wilmington, DE) and letrozole (Femara; Novartis, East Hanover, NJ), and the steroidal AI exemestane (Aromasin; Pharmacia, Peapack, NJ) have been associated with increased specificity and improved therapeutic index compared to nonselective AIs such as aminoglutethamide. Nonsteroidal and steroidal AIs have demonstrated to be superior to megestrol acetate in second-line therapy of postmenopausal women with metastatic breast cancer, and selective nonsteroidal AIs have shown to be superior to tamoxifen in first-line therapy of postmenopausal women with metastatic breast cancer. The ATAC (Arimidex, tamoxifen, alone, or in combination) trial is the only published randomized trial comparing the efficacy of an AI to tamoxifen for the adjuvant treatment of women with early breast cancer. This large

  4. New aromatase inhibitors. Synthesis and biological activity of aryl-substituted pyrrolizine and indolizine derivatives.

    PubMed

    Sonnet, P; Dallemagne, P; Guillon, J; Enguehard, C; Stiebing, S; Tanguy, J; Bureau, R; Rault, S; Auvray, P; Moslemi, S; Sourdaine, P; Séralini, G E

    2000-05-01

    We report herein the design and the synthesis of some aryl-substituted pyrrolizine and indolizine derivatives, on the basis of a hypothetical pharmacophore structure designed to fit the catalytic site of the human cytochrome P450 aromatase. The in vitro biological evaluation of these compounds allowed us to point out two new potent non-steroidal aromatase inhibitors, MR 20494 and MR 20492, with IC50 values in the range of 0.1 microM.

  5. Highly potent first examples of dual aromatase-steroid sulfatase inhibitors based on a biphenyl template.

    PubMed

    Woo, L W Lawrence; Jackson, Toby; Putey, Aurélien; Cozier, Gyles; Leonard, Philip; Acharya, K Ravi; Chander, Surinder K; Purohit, Atul; Reed, Michael J; Potter, Barry V L

    2010-03-11

    Single agents against multiple drug targets are of increasing interest. Hormone-dependent breast cancer (HDBC) may be more effectively treated by dual inhibition of aromatase and steroid sulfatase (STS). The aromatase inhibitory pharmacophore was thus introduced into a known biphenyl STS inhibitor to give a series of novel dual aromatase-sulfatase inhibitors (DASIs). Several compounds are good aromatase or STS inhibitors and DASI 20 (IC(50): aromatase, 2.0 nM; STS, 35 nM) and its chlorinated congener 23 (IC(50): aromatase, 0.5 nM; STS, 5.5 nM) are examples that show exceptional dual potency in JEG-3 cells. Both biphenyls share a para-sulfamate-containing ring B and a ring A, which contains a triazol-1-ylmethyl meta to the biphenyl bridge and para to a nitrile. At 1 mg/kg po, 20 and 23 reduced plasma estradiol levels strongly and inhibited liver STS activity potently in vivo. 23 is nonestrogenic and potently inhibits carbonic anhydrase II (IC(50) 86 nM). A complex was crystallized and its structure was solved by X-ray crystallography. This class of DASI should encourage further development toward multitargeted therapeutic intervention in HDBC.

  6. Two aromatase inhibitors inhibit the ability of a third to promote mating in male rats.

    PubMed

    Yahr, Pauline

    2015-09-01

    Aromatase, the enzyme that aromatizes androstenedione (A) to estrone and testosterone (T) to estradiol (E), affects androgen control of male sex behavior in many vertebrates. In male monkeys, rats and quail, E mimics the ability of T to promote mating, and aromatase inhibitors block mating induced by T but not E. Aromatase inhibitors include androgens with different A-rings than T and A, e.g., 1,4,6-androstatriene-3,17-dione (ATD), azoles, e.g., fadrozole, and androgens α-halogenated at carbon 6, e.g., 6α-bromoA, 6α-fluoroA and 6α-fluoroT. 6α-FluoroT is the only 6α-halogenated androgen studied in regard to mating. It promotes mating in male rats and quail and was studied, before it was known to inhibit aromatase, because it cannot be aromatized yet has the same A-ring as T. 6α-FluoroT might promote mating by binding estrogen receptors (ER) directly, i.e., unassisted, or by metabolism to an androgen that binds ER. Since neither process would require aromatase, this study tested both hypotheses by determining how mating induced in castrated male rats by 6α-fluoroT is affected by ATD and fadrozole. Both aromatase inhibitors inhibited the effects of 6α-fluoroT on mating. Thus, 6α-fluoroT does not promote mating by direct ER binding or metabolism to another androgen. Since aromatase underlies a process in which 6α-fluoroT, unlike most nonaromatizable androgens, mimics T effects on male sex behavior, the process must involve a feature that 6α-fluoroT shares with T but not other nonaromatizable androgens. A-ring structure is a candidate. A hypothesis is also offered for how aromatase may participate without aromatizing the androgen.

  7. Lichen sclerosus in a breast cancer survivor on an aromatase inhibitor: a case report.

    PubMed

    Potter, Jennifer E; Moore, Kendra A

    2013-04-01

    Lichen sclerosus is a commonly misdiagnosed disease that is characterized by thinned, hypopigmented, crinkled skin that often forms a figure-eight shape around the vaginal and anal openings. We present a case of advanced lichen sclerosus in a 53-year-old female patient prescribed a nonsteroidal aromatase inhibitor after the excision of a breast cancer tumor. We present a diagnostic approach to lichen sclerosus by recognizing its common figure-eight pattern, and we review the known causes and treatment of lichen sclerosus. Research has shown that lichen sclerosus is more common in low estrogen states, and thus it is logical that aromatase inhibitors could increase a patient's risk for developing this disease. We therefore propose that all patients prescribed aromatase inhibitors undergo regular vulvo-vaginal exams to rule out lichen sclerosus and other hypoestrogen-related vulvo-vaginal problems.

  8. Making males from females: the effects of aromatase inhibitors on a parthenogenetic species of whiptail lizard.

    PubMed

    Wennstrom, K L; Crews, D

    1995-09-01

    The parthenogenetic whiptail lizard Cnemidophorus uniparens provides a good model for the study of sex determination and sexual differentiation because genetic variation is minimal and all unmanipulated embryos will develop as females. Thus any deviation from the established course of development can be identified as a treatment effect. Previous work has shown that early prenatal treatment with CGS16949A, a nonsteroidal aromatase inhibitor, causes hatchlings to develop as males. The present study explores more fully the effects of dosage and timing of application of CGS16949A and examines the sex-reversing potential of CGS20267, a new and reputedly more potent aromatase inhibitor. Eggs were treated with a range of dosages of the aromatase inhibitors. Hatchlings that received 1 microgram or more of either inhibitor were all male, while those that received 0.1 microgram or less were all female. No difference in potency between the two compounds was detected. Animals treated with 100 micrograms of CGS16949A on Day 20 of incubation or later were all female, while those treated on Day 5 were all male. Seven sex-reversed male parthenogens have been raised to sexual maturity. The animals appear similar morphologically and behaviorally to males of the sexually reproducing whiptail species. Spermatogenesis and spermiogenesis have been confirmed by histological examination of the testes and by postcopulatory cloacal swabs. Application of aromatase inhibitors has been shown to sex-reverse both avian and reptilian species. In mammals, the male-determining gene of the Y chromosome (SRY) may code for an intrinsic aromatase inhibitor. Studies show the gene's product has a binding domain which recognizes regulatory elements in the promoter of the aromatase gene.(ABSTRACT TRUNCATED AT 250 WORDS)

  9. Aromatase, estrone sulfatase, and 17β-hydroxysteroid dehydrogenase: structure-function studies and inhibitor development.

    PubMed

    Hong, Yanyan; Chen, Shiuan

    2011-07-04

    Aromatase, estrone sulfatase, and 17β-hydroxysteroid dehydrogenase type 1 are involved in the key steps of 17β-estradiol biosynthesis. Structure-function studies of aromatase, estrone sulfatase and 17β-hydroxysteroid dehydrogenase type 1 are important to evaluate the molecular basis of the interaction between these enzymes and their inhibitors. Selective and potent inhibitors of the three enzymes have been developed as antiproliferative agents in hormone-dependent breast carcinoma. New treatment strategies for hormone-dependent breast cancer are discussed.

  10. Aromatase inhibitors as add-on treatment for men with epilepsy.

    PubMed

    Harden, Cynthia; MacLusky, Neil J

    2005-01-01

    Manipulation of neurosteroids to treat epilepsy has been an area of active research. The effect of testosterone on brain excitability and seizure threshold has been mixed; the estradiol metabolite of testosterone increases brain excitability, while the reduced metabolite of testosterone, 3alpha-androstanediol, decreases brain excitability, likely through an action at the gamma-amino butyric acid A receptor. Therefore, the metabolites of testosterone produce opposite effects on brain excitability in seizure models. Aromatase is the enzyme for the conversion of testosterone to 17beta-estradiol. Aromatase inhibitors could decrease brain excitability by decreasing local estradiol levels and therefore, could be beneficial for the treatment of epilepsy. Aromatase inhibitors are US Food and Drug Administration-approved and have a long history of safe use in menopausal women with breast cancer. This review presents the results of using anastrazole in an open-label, add-on manner in a small group of men with epilepsy in order to improve seizures. The results suggested some effect on reduction of seizures and no side effects. Testosterone levels did increase, but not to above the normal range. Letrozole used in a single case was also beneficial for seizures. It was concluded that aromatase inhibitors may be a useful adjunct to the treatment of epilepsy, but habituation to the treatment may be limiting. Many men with epilepsy have low testosterone, and aromatase inhibition may be helpful in restoring levels to normal. Modulation of reproductive hormones by aromatase inhibition as well as enhancement of the 3alpha-androstanediol pathway may be an avenue of epilepsy treatment that would not produce sedative side effects, which is often a limiting factor with standard antiseizure medications. A further interesting result is that elevated follicle stimulating hormone and luteal stimulating hormone levels were associated with seizure reduction, suggesting that they may be a

  11. Mechanisms of Aromatase Inhibitor-induced Musculoskeletal Symptoms

    DTIC Science & Technology

    2012-07-01

    neural physiology. For example, aromatase expression in the hippocampus protects hippocampal neurons against excitoxicity, which is thought to be a...Neuropharmacology 60, 580 (Mar, 2011). 32. I. H. Pang, M. R. Vasko, Morphine and norepinephrine but not 5-hydroxytryptamine and gamma- aminobutyric acid

  12. Tamoxifen metabolites as active inhibitors of aromatase in the treatment of breast cancer.

    PubMed

    Lu, Wenjie Jessie; Desta, Zeruesenay; Flockhart, David A

    2012-01-01

    The mechanism of tamoxifen action in the treatment of breast cancer is believed to be via active metabolites that act as potent estrogen receptor antagonists. Attempts to identify relationships between active metabolite concentrations and clinical outcomes have produced mixed results. Since anti-estrogenic effects may be brought about not only by estrogen antagonism, but also by reduced estrogen synthesis, we tested the ability of tamoxifen and its principal metabolites to inhibit aromatase in vitro. The activity of human aromatase in both recombinant and placental microsomal preparations was measured using the rate of generation of a fluorescent metabolite in the presence and absence of multiple concentrations of tamoxifen, endoxifen, N-desmethyl-tamoxifen, and Z-4-hydroxy-tamoxifen. Aromatase inhibition was further characterized by measuring the inhibition of testosterone metabolism to estradiol. The biochemical mechanisms of inhibition were documented and their inhibitory potency was compared. Using recombinant human aromatase, endoxifen, and N-desmethyl-tamoxifen were able to inhibit aromatase activity with K (i) values of 4.0 and 15.9 μM, respectively. Detailed characterization of inhibition by endoxifen and N-desmethyl-tamoxifen indicated non-competitive kinetics for both inhibitors. Similarly, endoxifen-inhibited testosterone metabolism via a non-competitive mechanism. No appreciable inhibition by tamoxifen or Z-4-hydroxy-tamoxifen was observed at similar concentrations. The relative inhibitory potency was: endoxifen > N-desmethyl-tamoxifen > Z-4-hydroxy-tamoxifen > tamoxifen. Similar data were obtained in human placental microsomes. Endoxifen and N-desmethyl-tamoxifen were found to be potent inhibitors of aromatase. Inhibition by these tamoxifen metabolites may contribute to the variability in clinical effects of tamoxifen in patients with breast cancer. Relationships between tamoxifen metabolite concentrations and clinical outcomes may be complex

  13. The vascular endothelial growth factor receptor inhibitor PTK787/ZK222584 inhibits aromatase.

    PubMed

    Banerjee, Susana; Zvelebil, Marketa; Furet, Pascal; Mueller-Vieira, Ursula; Evans, Dean B; Dowsett, Mitch; Martin, Lesley-Ann

    2009-06-01

    Endocrine therapy is well established for the treatment of breast cancer, and antiangiogenic agents are showing considerable promise. Targeting the vascular endothelial growth factor (VEGF) and estrogen receptor (ER) signaling pathways concomitantly may provide enhanced therapeutic benefit in ER-positive breast cancer. Therefore, the effects of the VEGF receptor (VEGFR) tyrosine kinase inhibitor PTK787/ZK222584 (PTK/ZK) were investigated using human breast cancer cell lines engineered to express aromatase. As expected in this system, estrogen (E2) or androstenedione induced a proliferative response and increased ER-mediated transcription in ER-positive cell lines expressing aromatase. However, surprisingly, in the presence of androstenedione, PTK/ZK suppressed both the androstenedione-stimulated proliferation and ER-mediated transcription. PTK/ZK alone and in the presence of E2 had no observable effect on proliferation or ER-mediated transcription. These effects result from PTK/ZK having previously unrecognized antiaromatase activity and PTK/ZK being a competitive aromatase inhibitor. Computer-assisted molecular modeling showed that PTK/ZK could potentially bind directly to aromatase. The demonstration that PTK/ZK inhibits aromatase and VEGFR indicates that agents cross-inhibiting two important classes of targets in breast cancer could be developed.

  14. Novel inhibitor discovery against aromatase through virtual screening and molecular dynamic simulation: a computational approach in drug design.

    PubMed

    Mirzaie, Sako; Chupani, Latifeh; Asadabadi, Ebrahim Barzegari; Shahverdi, Ahmad Reza; Jamalan, Mostafa

    2013-01-01

    Inhibition of aromatase (CYTP450) as a key enzyme in the estrogen biosynthesis could result in regression of estrogen-dependent tumors and even preventing the promotion of breast cancer. Although today potent steroid and non-steroid inhibitors of aromatase are available, isoflavanone derivatives as natural compounds with least side effects have been described as the candidate for a new generation of aromatase inhibitors. 2a as an isoflavanone derivative is the most potent inhibitor of aromatase, synthesized by Bonfield et al. (2012[7]). In our computational study, the mentioned compound was used as the template for virtual screening. Between 286 selected compounds with 70 % of structural similarity to 2a, 150 of them showed lower docking energy in comparison with 2a. Compound 2a_1 with 11.2 kcal/mol had the lowest docking energy. Interaction of 2a_1 with aromatase was further investigated and compared with 2a and androstenedione (ASD) as a natural substrate of aromatase, through 20 ns of molecular dynamic simulation. Analysis of trajectories showed, while ASD interacts with aromatase through hydrogen bonds and 2a just interacts via hydrophobic forces, 2a_1 not only accommodates in the hydrophobic active site of aromatase in a suitable manner but it also makes a stable coordination with iron atom of aromatase heme group via OB.

  15. Direct Effects, Compensation, and Recovery in Female Fathead Minnows Exposed to a Model Aromatase Inhibitor

    EPA Science Inventory

    The paper reports on the effects of a model aromatase inhibitor, fadrozole, on molecular and biochemical endpoints within the fathead minnow reproductive axis. Unlike previous studies, this work incorporated extensive time-course characterization over the course of an 8 d exposu...

  16. A Study to Evaluate Genetic Predictors of Aromatase Inhibitor Musculoskeletal Symptoms (AIMSS) | Division of Cancer Prevention

    Cancer.gov

    E1Z11 is a study to determine whether certain genetic information can predict which breast cancer patients will discontinue treatment with AIs due to the development of musculoskeletal symptoms (MSS). Women with stage 1-111 breast cancer who are prescribed the aromatase inhibitor anastrozole as treatment may join. |

  17. Synthesis of aromatase inhibitors and dual aromatase steroid sulfatase inhibitors by linking an arylsulfamate motif to 4-(4H-1,2,4-triazol-4-ylamino)benzonitrile: SAR, crystal structures, in vitro and in vivo activities.

    PubMed

    Bubert, Christian; Woo, L W Lawrence; Sutcliffe, Oliver B; Mahon, Mary F; Chander, Surinder K; Purohit, Atul; Reed, Michael J; Potter, Barry V L

    2008-11-01

    4-(((4-Cyanophenyl)(4H-1,2,4-triazol-4-yl)amino)methyl)phenyl sulfamate (6 a) was the first dual aromatase-sulfatase inhibitor (DASI) reported. Several series of its derivatives with various linker systems between the steroid sulfatase (STS) and the aromatase inhibitory pharmacophores were synthesised and evaluated in JEG-3 cells. The X-ray crystal structures of the aromatase inhibitors, DASI precursors 42 d and 60, and DASI 43 h were determined. Nearly all derivatives show improved in vitro aromatase inhibition over 6 a but decreased STS inhibition. The best aromatase inhibitor is 42 e (IC(50)=0.26 nM) and the best DASI is 43 e (IC(50 aromatase)=0.45 nM, IC(50 STS)=1200 nM). SAR for aromatase inhibition shows that compounds containing an alkylene- and thioether-based linker system are more potent than those that are ether-, sulfone-, or sulfonamide-based, and that the length of the linker has a limited effect on aromatase inhibition beyond two methylene units. Compounds 43 d-f were studied in vivo (10 mg kg(-1), single, p.o.). The most potent DASI is 43 e, which inhibited PMSG-induced plasma estradiol levels by 92 % and liver STS activity by 98 % 3 h after dosing. These results further strengthen the concept of designing and developing DASIs for potential treatment of hormone-related cancers.

  18. Design and synthesis of a new type of non steroidal human aromatase inhibitors.

    PubMed

    Sonnet, P; Guillon, J; Enguehard, C; Dallemagne, P; Bureau, R; Rault S Auvray, P; Moslemi, S; Sourdiane, P; Galopin, S; Séralini, G E

    1998-05-05

    The structure-activity relationship study of one of recently described aromatase inhibitors, compound 1 (MR20814), allowed us to design some related derivatives as potential new inhibitors. Among those we synthesized, chlorophenylpyridylmethylenetetrahydroindolizinone 5 (MR20492) exhibited in vitro a ten-fold higher inhibition of the enzyme (IC50 = 0.2 +/- 0.0 microM and Ki = 10.3 +/- 3.3 nM).

  19. Development of a new class of aromatase inhibitors: Design, synthesis and inhibitory activity of 3-phenylchroman-4-one (isoflavanone) derivatives

    PubMed Central

    Bonfield, Kevin; Amato, Erica; Bankemper, Tony; Agard, Hannah; Steller, Jeffrey; Keeler, James M.; Roy, David; McCallum, Adam; Paula, Stefan; Ma, Lili

    2014-01-01

    Aromatase (CYP19) catalyzes the aromatization reaction of androgen substrates to estrogens, the last and rate-limiting step in estrogen biosynthesis. Inhibition of aromatase is a new and promising approach to treat hormone-dependent breast cancer. We present here the design and development of isoflavanone derivatives as potential aromatase inhibitors. Structural modifications were performed on the A and B rings of isoflavanones via microwave-assisted, gold-catalyzed annulation reactions of hydroxyaldehydes and alkynes. The in vitro aromatase inhibition of these compounds was determined by fluorescence-based assays utilizing recombinant human aromatase (baculovirus/insect cell-expressed). The compounds 3-(4-phenoxyphenyl)chroman-4-one (1h), 6-methoxy-3-phenylchroman-4-one (2a) and 3-(pyridin-3-yl)chroman-4-one (3b) exhibited potent inhibitory effects against aromatase with IC50 values of 2.4 μM, 0.26 μM and 5.8 μM, respectively. Docking simulations were employed to investigate crucial enzyme/inhibitor interactions such as hydrophobic interactions, hydrogen bonding and heme iron coordination. This report provides useful information on aromatase inhibition and serves as a starting point for the development of new flavonoid aromatase inhibitors. PMID:22444875

  20. Isolation and Characterization of Aromatase Inhibitors from Brassaiopsis glomerulata (Araliaceae).

    PubMed

    Balunas, Marcy J; Su, Bin; Riswan, Soedarsono; Fong, Harry H S; Brueggemeier, Robert W; Pezzuto, John M; Kinghorn, A Douglas

    2009-02-19

    The hexane- and ethyl acetate-soluble extracts of the leaves of Brassaiopsis glomerulata (Blume) Regel (Araliaceae), collected in Indonesia, were found to inhibit aromatase, the rate-limiting enzyme in the production of estrogens from androgens, in both enzyme- and cell-based aromatase inhibition (AI) assays. Bioassay-guided fractionation led to the isolation of six known compounds of the steroid and triterpenoid classes (1-6) from the hexane extract, of which 6β-hydroxystimasta-4-en-3-one (5), was moderately active in the cell-based AI assay. Fractionation of the ethyl acetate extract afforded seven pure isolates (7-13) of the modified peptide, fatty acid, monoterpenoid, and benzenoid types, including six known compounds and the new natural product, N-benzoyl-L-phenylalanine methyl ester (9). The absolute stereochemistry of 9 and the other two peptides, 7 and 8, was determined by Marfey's analysis. Linoleic acid (10) was found to be active in the enzyme-based AI assay, while 9 and (-)-dehydrololiolide (12) showed activity in the cell-based AI assay.

  1. Aromatase inhibitors: mechanism of action and role in the treatment of breast cancer.

    PubMed

    Miller, William R

    2003-08-01

    The natural history of breast cancer is closely linked with estrogens. These hormones influence both risk to the disease and growth of many established tumors. Consequently, measures that either inhibit the synthesis or block the mechanism of action of estrogens are attractive strategies for therapeutic intervention. This is particularly true in postmenopausal women in whom hormone responsiveness is common and estrogen synthesis is primarily sited peripherally in adipose tissue, muscle and breast tissue, rather than in the ovaries as in premenopausal women. In terms of inhibiting production, the most specific effects are best achieved by blocking the last step in biosynthesis, the conversion of androgens to estrogens by the heme-containing enzyme, aromatase. Two major classes of aromatase inhibitors have been developed and are currently in clinical use. Type I steroidal drugs include formestane and exemestane; they are androgen substrate analogues that bind competitively but irreversibly to the enzyme and have been marketed as "inactivators." Type II nonsteroidal inhibitors such as anastrozole and letrozole are triazoles; they bind reversibly to the enzyme and fit into the substrate binding site, such that azole nitrogens interact with the heme prosthetic group. This type of association provides exquisite potency for and specificity against the aromatase enzyme. These agents represent several generations of development, with each step in the evolution producing an increase in both potency and specificity. The latest aromatase inhibitors are drugs of immense potential that will undoubtedly play a major role in the management of postmenopausal women with hormone-dependent breast cancer. They also represent tools by which to elucidate the roles of both aromatase and estrogen in the development and growth of breast cancer.

  2. ALTERATIONS IN THE TRANSCRIPTOME AND PROTEOME OF ZEBRAFISH (DANIO RERIO) EXPOSED TO FADROZOLE, A MODEL AROMATASE INHIBITOR

    EPA Science Inventory

    Fadrozole is a reversible, competitive inhibitor of aromatase activity and therefore an endocrine-disrupting compound (EDC) that disrupts steroidogenesis by inhibiting the conversion of testosterone to 172-estradiol. While fadrozole is a therapeutic drug with generally no enviro...

  3. Benefit/risk for adjuvant breast cancer therapy with tamoxifen or aromatase inhibitor use by age, and race/ethnicity.

    PubMed

    Chlebowski, R T; Haque, R; Hedlin, H; Col, N; Paskett, E; Manson, J E; Kubo, J T; Johnson, K C; Wactawski-Wende, J; Pan, K; Anderson, G

    2015-12-01

    In early adjuvant breast cancer trial reports, aromatase inhibitors more effectively reduced breast recurrence with lower risk of thromboembolic events and endometrial cancer than tamoxifen, while aromatase inhibitors had higher fracture and cardiovascular disease risk. We used data from updated patient-level meta-analyses of adjuvant trials in analyses to summarize the benefits and risks of these agents in various clinical circumstances. Baseline incidence rates for health outcomes by age and race/ethnicity, absent aromatase inhibitor, or tamoxifen use were estimated from the Women's Health Initiative. Aromatase inhibitor and tamoxifen effects on distant recurrence were obtained from a meta-analysis of the Arimidex, Tamoxifen, Alone or in Combination (ATAC) and Breast International Group (Big-1-98) clinical trials. Impact on other health outcomes were obtained from meta-analyses of randomized trials comparing aromatase inhibitor to tamoxifen use and from placebo-controlled chemoprevention trials. All health outcomes were given equal weight when modeling net benefit/risk for aromatase inhibitor compared to tamoxifen use by breast cancer recurrence risk, age (decade), race/ethnicity, hysterectomy (yes/no), and by prior myocardial infarction. Over a 10-year period, the benefit/risk index was more favorable for aromatase inhibitor than for tamoxifen as adjuvant breast cancer therapy in almost all circumstances regardless of patient age, race/ethnicity, breast cancer recurrence risk, or presence or absence of a uterus. Only in older women with prior myocardial infarction and low recurrence risk was an advantage for tamoxifen seen. Using a benefit/risk index for endocrine adjuvant breast cancer therapy in postmenopausal women, benefit was higher for aromatase inhibitor use in almost all circumstances.

  4. Effects of aromatase inhibitor on menopausal hyperplasia in a case of obesity.

    PubMed

    Koloszar, S; Pal, Z; Kereszturi, A; Vajda, G; Pal, A; Daru, J

    2012-02-01

    The aromatase inhibitor anastrazole proved effective in the treatment of endometrial hyperplasia and postmenopausal bleeding in an obese 65-year-old woman with high operative risk. During anastrazole administration for 12 months, the endometrial thickness decreased from 9.8 mm to 2.4 mm and the control endometrial histology showed an atrophic endometrium. Uterine bleeding did not occur in the post-treatment, 3-year follow-up period. The endometrial thicknesses measured yearly by ultrasonography were 2.9, 3.5 and 3.3 mm. The plasma estradiol levels increased from < 73 pmol/l post-treatment to 112, 98 and 103 pmol/l. This case demonstrates that long-term aromatase inhibitor treatment can result in a refractory status of the endometrium and the estradiol produced in the adipose tissue does not exert a proliferative effect.

  5. Efficacy and mechanism of action of Proellex, an antiprogestin in aromatase overexpressing and Letrozole resistant T47D breast cancer cells.

    PubMed

    Gupta, Akash; Mehta, Rajeshwari; Alimirah, Fatouma; Peng, Xinjian; Murillo, Genoveva; Wiehle, Ronald; Mehta, Rajendra G

    2013-01-01

    Aromatase inhibitors (AI) are considered as a first line therapy for ER+PR+ breast cancers. However, many patients acquire resistance to AI. In this study, we determined the response of antiprogestin CDB-4124 (Proellex) on the aromatase overexpressing and Letrozole resistant cell lines and also studies its mechanism of action in inhibition of breast cancer cell proliferation. For these studies we generated aromatase overexpressing T47D (T47Darom) and respective control (T47Dcon) breast cancer cell lines by stable transfection with plasmid containing CYP19A1 gene, or empty vector respectively. Letrozole resistant cell line (T47DaromLR) was generated by incubating T47Darom for 75 weeks in the presence of 10 μM Letrozole. Cell proliferation was determined by MTT or crystal violet assays. Gene expressions were quantified by QRT-PCR whereas proteins were identified by western blot analyses, flow cytometry and immunofluorescence staining. Aromatase activity was determined by estradiol ELISA. The effects of Proellex on the anchorage independent growth were measured by soft agar colony formation. Statistical differences between the various groups were determined by Student's 't' test or ANOVA followed by Bonferroni's post hoc test. Results showed that T47Darom and T47DaromLR cell lines had significantly higher aromatase expression (mRNA; 80-90 fold and protein) and as a result exhibited increased aromatization of testosterone to estradiol as compared to T47Dcon. Both these cell lines showed enhanced growth in the presence of Testosterone (50-60%). In T47DaromLR cells increased PR-B and EGFR expression as compared to T47Dcon cells was observed. Proellex and other known aromatase inhibitors (Letrozole, Anastrozole, and Exemestane) inhibited testosterone induced cell proliferation and anchorage independent growth of T47Darom cells. Cell growth inhibition was significantly greater when cells were treated with Proellex alone or in combination with other AIs as compared to AIs

  6. Exercise intervention in breast cancer patients with aromatase inhibitor-associated arthralgia: a pilot study.

    PubMed

    DeNysschen, C A; Burton, H; Ademuyiwa, F; Levine, E; Tetewsky, S; O'Connor, T

    2014-07-01

    Aromatase inhibitors (AIs) block estrogen synthesis and are commonly used as adjuvant treatments for breast cancer patients. A common side effect is joint pain. This was a pilot study to examine implementation of an exercise program in reducing joint pain and improving quality of life (QoL) and functional performance in breast cancer patients treated with AIs. Twenty-six participants completed an 8-week, home-based program that combined upper and lower body resistance exercises with self-selected aerobic exercises. We measured: (1) anthropometry (2) functional performance (grip strength, biceps curl to exhaustion, and sit-to-stand and cardiovascular endurance (3-min step test). Joint pain and QoL were assessed using self-administered surveys. Participants reported a significantly lower number of painful joints, an improvement in QoL and a reduction in depressive symptoms. Significant improvements in grip strength, biceps curl, and sit-to-stand (by 14%, 51% and 15% respectively) were also observed. However, we found no significant changes in cardiovascular endurance or in anthropometric measures. An 8-week, home-based exercise program may provide potential benefit to the breast cancer patients undergoing AI treatment by reducing joint pain, improving functional performance and QoL, and reducing depressive symptoms. Further studies are needed to confirm these results.

  7. Mechanism-based categorization of aromatase inhibitors: a potential discovery and screening tool.

    PubMed

    Petkov, P I; Temelkov, S; Villeneuve, D L; Ankley, G T; Mekenyan, O G

    2009-10-01

    Cytochrome P450 aromatase is a key steroidogenic enzyme that converts androgens to estrogens in vertebrates. There is much interest in aromatase inhibitors (AIs) both because of their use as pharmaceuticals in the treatment of estrogen-sensitive breast cancers, and because a number of environmental contaminants can act as AIs, thereby disrupting endocrine function in humans and wildlife through suppression of circulating estrogen levels. The goal of the current work was to develop a mechanism-based structure-activity relationship (SAR) categorization framework highlighting the most important chemical structural features responsible for inhibition of aromatase activity. Two main interaction mechanisms were discerned: steroidal and non-steroidal. The steroid scaffold is most prominent when the structure of the target chemical is similar to the natural substrates of aromatase - androstenedione and testosterone. Chemicals acting by non-steroidal mechanism(s) possess a heteroatom (N, O, S) able to coordinate the heme iron of the cytochrome P450, and thus interfere with steroid hydroxylation. The specific structural boundaries controlling AI for both analyzed mechanisms were defined, and a software tool was developed that allowed a decision tree (profile) to be built discriminating AIs by mechanism and potency. An input chemical follows a profiling path and the structure is examined at each step to decide whether it conforms with the structural boundaries implemented in the decision tree node. Such a system would aid drug discovery efforts, as well as provide a screening tool to detect environmental contaminants that could act as AIs.

  8. Functional genetic polymorphisms in the aromatase gene CYP19 vary the response of breast cancer patients to neoadjuvant therapy with aromatase inhibitors.

    PubMed

    Wang, Liewei; Ellsworth, Katarzyna A; Moon, Irene; Pelleymounter, Linda L; Eckloff, Bruce W; Martin, Yvette N; Fridley, Brooke L; Jenkins, Gregory D; Batzler, Anthony; Suman, Vera J; Ravi, Saranya; Dixon, J Michael; Miller, William R; Wieben, Eric D; Buzdar, Aman; Weinshilboum, Richard M; Ingle, James N

    2010-01-01

    Aromatase (CYP19) is a critical enzyme in estrogen biosynthesis and aromatase inhibitors (AI) are employed widely for endocrine therapy in postmenopausal women with breast cancer. We hypothesized that single nucleotide polymorphisms (SNPs) in the CYP19 gene may alter the effectiveness of AI therapy in the neoadjuvant setting. Genomic DNA was obtained for sequencing from 52 women pre-AI and post-AI treatment in this setting. Additionally, genomic DNA obtained from 82 samples of breast cancer and 19 samples of normal breast tissue was subjected to resequencing. No differences in CYP19 sequence were observed between tumor and germ-line DNA in the same patient. A total of 48 SNPs were identified including 4 novel SNPs when compared with previous resequencing data. For genotype-phenotype association studies, we determined the levels of aromatase activity, estrone, estradiol, and tumor size in patients pre-AI and post-AI treatment. We defined two tightly linked SNPs (rs6493497 and rs7176005 in the 5'-flanking region of CYP19 exon 1.1) that were significantly associated with a greater change in aromatase activity after AI treatment. In a follow-up study of 200 women with early-stage breast cancer who were treated with adjuvant anastrozole, these same two SNPs were also associated with higher plasma estradiol levels in patients pre-AI and post-AI treatment. Electrophoretic mobility shift and reporter gene assays confirmed likely functional effects of these two SNPs on transcription of CYP19. Our findings indicate that two common genetic polymorphisms in the aromatase gene CYP19 vary the response of breast cancer patients to aromatase inhibitors.

  9. Adjuvant therapy with tamoxifen compared to aromatase inhibitors for 257 male breast cancer patients.

    PubMed

    Eggemann, Holm; Ignatov, Atanas; Smith, Bobbie J; Altmann, Udo; von Minckwitz, Gunter; Röhl, Freidrich W; Jahn, Mark; Costa, Serban-Dan

    2013-01-01

    To determine the impact of adjuvant treatment with tamoxifen and aromatase inhibitors (AI) on the survival of men with breast cancer. We analyzed 257 male patients with hormone-receptor-positive breast cancer from numerous German population-based cancer registries treated with tamoxifen (N = 207) or aromatase inhibitors (N = 50). The median follow-up was 42.2 (range 2-115) months. Median age at diagnosis was 68 (range 36-91) years. Thirty-seven (17.9 %) patients treated with tamoxifen and 16 (32.0 %) patients treated with AI died (log rank p = 0.007). After the adjustment for the patient's age, tumor size, node status, and tumor grading, the AI treatment was linked to a 1.5-fold increase in risk of mortality compared to tamoxifen (HR 1.55; 95 % CI: 1.13-2.13; p = 0.007). The overall survival in male breast cancer was significantly better after adjuvant treatment with tamoxifen compared to an aromatase inhibitor. Tamoxifen should be considered as the treatment of choice for hormone-receptor-positive male breast cancer.

  10. Steroidal inhibitors as chemical probes of the active site of aromatase.

    PubMed

    Brueggemeir, R W; Moh, P P; Ebrahimian, S; Darby, M V

    1993-03-01

    Androstenedione analogs containing 7 alpha-substituents have proven to be potent inhibitors of aromatase in human placental microsomes, in MCF-7 mammary cell cultures, and in JAr choriocarcinoma cells. Recent investigations have focused on the use of mechanism-based inhibitors, such as 7 alpha-substituted 1,4-androstadienediones, to biochemically probe the active site of aromatase. Inhibition kinetics were determined under initial velocity conditions using purified human placental cytochrome P450arom protein in a reconstituted system. Derivatives of 1,4-androstadiene-3,17-dione and 1,4,6-androstatriene-3,17-dione exhibited high affinity in the purified enzyme system. 7 alpha-(4'-Amino)phenylthio-1,4-androstadiene-3,17-dione, abbreviated 7 alpha-APTADD, demonstrated rapid time-dependent, first-order inactivation of reconstituted aromatase activity only in the presence of NADPH. The apparent Kinact for 7 alpha-APTADD is 11.8 nM, the first-order rate of inactivation is 2.72 x 10(-3) sec-1, and the half-time of inactivation at infinite inhibitor concentration is 4.25 min. The values for the rate constant and half-time of inactivation are similar to those observed in the placental microsomal assay system. Further studies were performed with radioiodinated 7 alpha-(4'-iodo)phenylthio-1,4-androstadienedione, 7 alpha-IPTADD, and the reconstituted aromatase system. Incubations with [125I] 7 alpha-IPTADD were followed by protein precipitation, solvent extraction, and column chromatography. Analysis of the isolated cytochrome P450arom by gel electrophoresis and autoradiography demonstrated the presence of only one radioactive band, which corresponded to the protein staining band for cytochrome P450arom. HPLC radiochromatographic analysis of the isolated cytochrome P450aroM confirmed the presence of only one radioactive peak coeluting with the u.v. peak for cytochrome P450arom. Peptide mapping analysis by reverse-phase HPLC of digested inhibitor-cytochrome P450arom complex

  11. Coadministrating luteolin minimizes the side effects of the aromatase inhibitor letrozole.

    PubMed

    Li, Fengjuan; Wong, Tsz Yan; Lin, Shu-mei; Chow, Simon; Cheung, Wing-hoi; Chan, Franky L; Chen, Shiuan; Leung, Lai K

    2014-11-01

    Aromatase inhibitors (AIs) have been used as adjuvant therapeutic agents for breast cancer. Their adverse side effect on blood lipid is well documented. Some natural compounds have been shown to be potential AIs. In the present study, we compared the efficacy of the flavonoid luteolin to the clinically approved AI letrozole (Femara; Novartis Pharmaceuticals, East Hanover, NJ) in a cell and a mouse model. In the in vitro experimental results for aromatase inhibition, the Ki values of luteolin and letrozole were estimated to be 2.44 µM and 0.41 nM, respectively. Both letrozole and luteolin appeared to be competitive inhibitors. Subsequently, an animal model was used for the comparison. Aromatase-expressing MCF-7 cells were transplanted into ovariectomized athymic mice. Luteolin was given by mouth at 5, 20, and 50 mg/kg, whereas letrozole was administered by intravenous injection. Similar to letrozole, luteolin administration reduced plasma estrogen concentrations and suppressed the xenograft proliferation. The regulation of cell cycle and apoptotic proteins-such as a decrease in the expression of Bcl-xL, cyclin-A/D1/E, CDK2/4, and increase in that of Bax-was about the same in both treatments. The most significant disparity was on blood lipids. In contrast to letrozole, luteolin increased fasting plasma high-density lipoprotein concentrations and produced a desirable blood lipid profile. These results suggested that the flavonoid could be a coadjuvant therapeutic agent without impairing the action of AIs.

  12. Design, synthesis, and biological evaluation of resveratrol analogues as aromatase and quinone reductase 2 inhibitors for chemoprevention of cancer.

    PubMed

    Sun, Bin; Hoshino, Juma; Jermihov, Katie; Marler, Laura; Pezzuto, John M; Mesecar, Andrew D; Cushman, Mark

    2010-07-15

    A series of new resveratrol analogues were designed and synthesized and their inhibitory activities against aromatase were evaluated. The crystal structure of human aromatase (PDB 3eqm) was used to rationalize the mechanism of action of the aromatase inhibitor 32 (IC50 0.59 microM) through docking, molecular mechanics energy minimization, and computer graphics molecular modeling, and the information was utilized to design several very potent inhibitors, including compounds 82 (IC50 70 nM) and 84 (IC50 36 nM). The aromatase inhibitory activities of these compounds are much more potent than that for the lead compound resveratrol, which has an IC50 of 80 microM. In addition to aromatase inhibitory activity, compounds 32 and 44 also displayed potent QR2 inhibitory activity (IC50 1.7 microM and 0.27 microM, respectively) and the high-resolution X-ray structures of QR2 in complex with these two compounds provide insight into their mechanism of QR2 inhibition. The aromatase and quinone reductase inhibitors resulting from these studies have potential value in the treatment and prevention of cancer.

  13. Design, synthesis, and biological evaluation of resveratrol analogues as aromatase and quinone reductase 2 inhibitors for chemoprevention of cancer

    SciTech Connect

    Sun, Bin; Hoshino, Juma; Jermihov, Katie; Marler, Laura; Pezzuto, John M.; Mesecar, Andrew D.; Cushman, Mark

    2012-07-11

    A series of new resveratrol analogues were designed and synthesized and their inhibitory activities against aromatase were evaluated. The crystal structure of human aromatase (PDB 3eqm) was used to rationalize the mechanism of action of the aromatase inhibitor 32 (IC{sub 50} 0.59 {mu}M) through docking, molecular mechanics energy minimization, and computer graphics molecular modeling, and the information was utilized to design several very potent inhibitors, including compounds 82 (IC{sub 50} 70 nM) and 84 (IC{sub 50} 36 nM). The aromatase inhibitory activities of these compounds are much more potent than that for the lead compound resveratrol, which has an IC{sub 50} of 80 {mu}M. In addition to aromatase inhibitory activity, compounds 32 and 44 also displayed potent QR2 inhibitory activity (IC{sub 50} 1.7 {mu}M and 0.27 {mu}M, respectively) and the high-resolution X-ray structures of QR2 in complex with these two compounds provide insight into their mechanism of QR2 inhibition. The aromatase and quinone reductase inhibitors resulting from these studies have potential value in the treatment and prevention of cancer.

  14. Synthesis of Triphenylethylene Bisphenols as Aromatase Inhibitors that Also Modulate Estrogen Receptors

    PubMed Central

    Lv, Wei; Liu, Jinzhong; Skaar, Todd C.; O'Neill, Elizaveta; Yu, Ge; Flockhart, David A.; Cushman, Mark

    2016-01-01

    A series of triphenylethylene bisphenol analogues of the selective estrogen receptor modulator (SERM) tamoxifen were synthesized and evaluated for their abilities to inhibit aromatase, bind to estrogen receptor-α (ER-α) and estrogen receptor-β (ER-β), and antagonize the activity of β-estradiol in MCF-7 human breast cancer cells. The long-range goal has been to create dual aromatase inhibitor (AI)/selective estrogen receptor modulators (SERMs). The hypothesis is that in normal tissue the estrogenic SERM activity of a dual AI/SERM could attenuate the undesired effects stemming from global estrogen depletion caused by the AI activity of a dual AI/SERM, while in breast cancer tissue the antiestrogenic SERM activity of a dual AI/SERM could act synergistically with AI activity to enhance the antiproliferative effect. The potent aromatase inhibitory activities and high ER-α and ER-β binding affinities of several of the resulting analogues, together with the facts that they antagonize β-estradiol in a functional assay in MCF-7 human breast cancer cells and they have no E/Z isomers, support their further development in order to obtain dual AI/SERM agents for breast cancer treatment. PMID:26704594

  15. Efficacy of Exemestane in Korean Patients with Metastatic Breast Cancer after Failure of Nonsteroidal Aromatase Inhibitors

    PubMed Central

    Lee, June Koo; Lee, Daewon; Kim, Ji-Yeon; Lim, Yoojoo; Lee, Eunyoung; Moon, Hyeong-Gon; Kim, Tae-Yong; Han, Sae-Won; Oh, Do-Youn; Lee, Se-Hoon; Han, Wonshik; Kim, Dong-Wan; Kim, Tae-You; Noh, Dong-Young

    2013-01-01

    Purpose Exemestane has shown good efficacy and tolerability in postmenopausal women with hormone receptor-positive metastatic breast cancer. However, clinical outcomes in Korean patients have not yet been reported. Methods Data on 112 postmenopausal women with metastatic breast cancer were obtained retrospectively. Clinicopathological characteristics and treatment history were extracted from medical records. All patients received 25 mg exemestane daily until objective disease progression. Progression-free survival (PFS) was the primary endpoint, and secondary endpoints were overall survival (OS), objective response rate (ORR), and clinical benefit rate (CBR=complete response+partial response+stable disease for 6 months). Results The median age of the subjects was 55 years (range, 28-76 years). Exemestane treatment resulted in a median PFS of 5.7 months (95% confidence interval [CI], 4.4-7.0 months) and median OS of 21.9 months (95% CI, 13.6-30.3 months). ORR was 6.4% and CBR was 46.4% for the 110 patients with evaluable lesions. Symptomatic visceral disease was independently associated with shorter PFS (hazard ratio, 3.611; 95% CI, 1.904-6.848; p<0.001), compared with bone-dominant disease in a multivariate analysis of PFS after adjusting for age, hormone receptor, human epidermal growth factor receptor 2, Ki-67 status, dominant metastasis site, and sensitivity to nonsteroidal aromatase inhibitor (AI) treatment. Sensitivity to previous nonsteroidal AI treatment was not associated with PFS, suggesting no cross-resistance between exemestane and nonsteroidal AIs. Conclusion Exemestane was effective in postmenopausal Korean women with hormone receptor-positive metastatic breast cancer who failed previous nonsteroidal AI treatment. PMID:23593084

  16. The aromatase inhibitors (plus ovarian function suppression) in premenopausal breast cancer patients: ready for prime time?

    PubMed

    Montagna, Emilia; Cancello, Giuseppe; Colleoni, Marco

    2013-12-01

    Tamoxifen alone or the combination of ovarian function suppression (OFS) and tamoxifen are the mainstay of hormonal therapy in premenopausal women with endocrine-responsive breast cancer. The results of large trials conducted with the third generation of aromatase inhibitors (AIs) in the metastatic, neoadjuvant and adjuvant setting, indicated better outcomes among postmenopausal breast cancer patients with endocrine responsive disease given AIs than among those given tamoxifen. These results supported the investigation of AIs in combination with OFS in premenopausal women with hormone receptor positive breast cancer. In this article we reviewed the efficacy and toxicity data on the use of AIs combined with OFS in premenopausal breast cancer patients in metastatic, neoadjuvant and adjuvant setting. Given the available evidence at the time in metastatic setting for premenopausal patients suitable of endocrine therapy the AI is a viable option, if tamoxifen resistance is proven, although mandates the use of OFS. In neoadjuvant setting the AIs in combination of OFS should not be used outside of a clinical trial. In the adjuvant setting, tamoxifen alone or OFS plus tamoxifen are reasonable options. Despite the lack of conclusive data favoring the combination of tamoxifen plus OFS, this treatment might be a reasonable option for subgroups of patients such as very young patients, OFS alone should nort be considered unless tamoxifen was contraindicated. Similarly, in cases where tamoxifen is contraindicated, AIs as an adjunct to OFS is a treatment option in premenopausal patients. New large randomized studies are required to confirm the role of OFS plus an AI in premenopausal women.

  17. Aromatase Inhibitors for Endometriosis-Associated Infertility; Do We Have Sufficient Evidence?

    PubMed Central

    Abu Hashim, Hatem

    2016-01-01

    Orally active aromatase inhibitors (AIs) have gained attention for treatment of infertile women with endometriosis in whom aromatase p450 is aberrantly expressed. This review aimed to critically appraise and summarize the available evidence concerning the use of AIs for management of endometriosis-associated infertility. PubMed was searched to May 2015 with the following key words: endometriosis, infertility and aromatase. Priority was given for randomized controlled trials (RCTs) followed by other study designs. Main outcome measures were as follows: rates of clinical pregnancy, miscarriage and live birth as well as endocrine outcomes. Eighty-two abstracts were screened and six original articles were included. A RCT demonstrated that post-operative letrozole treatment did not improve spontaneous pregnancy rate. Another RCT reported no superiority of letrozole superovulation over clomiphene citrate (each combined with intrauterine insemination) in minimalmild endometriosis and previous laparoscopic treatment. Anastrozole significantly inhibited the growth of endometriotic cells and their estrogen production in culture. In assisted reproductive technology (ART) cycles, dual suppression (Agonist/anastrozole) was tested in a pilot study with a pregnancy rate of 45% however, high pregnancy loss (30%) occurred. A retrospective study showed that letrozole may improve endometrial receptivity in endometriotic patients undergoing in vitro fertilization (IVF). An opposite view from an in vitro study showed lower estradiol production and aromatase expression in cultured granulosa cells from endometriotic women undergoing IVF and marked reduction under letrozole. In conclusion, current evidence is limited. More trials are warranted to enhance our knowledge and provide a clear and unequivocal evidence to guide our clinical management of infertile women with endometriosis using AIs. PMID:27695608

  18. Methadone: a substrate and mechanism-based inhibitor of CYP19 (aromatase).

    PubMed

    Lu, Wenjie Jessie; Bies, Robert; Kamden, Landry K; Desta, Zeruesenay; Flockhart, David A

    2010-08-01

    The peripheral conversion of testosterone to estradiol by aromatase is the primary source of endogenous estrogen in postmenopausal women. Studies indicating that placental aromatase is able to metabolize methadone to its primary metabolite, 2-ethylidene-1, 5-dimethyl-3, 3-diphenylpyrrolidin (EDDP), led us to test the hypothesis that methadone is able to act as an inhibitor of aromatase. Using recombinant human CYP19, we examined the ability of methadone to bring about either reversible or mechanism-based inhibition of the conversion of testosterone to estradiol. To test for reversible inhibition, racemic methadone or its metabolite EDDP or 2-ethyl-5-methyl-3, 3-diphenylpyrroline (EMDP) was incubated for 30 min with testosterone at the K(m) (4 microM). To test for mechanism-based inhibition, microsomal preincubations were performed for up to 30 min using racemic methadone (1-1000 microM), R- or S-methadone (0.5-500 microM), or EDDP or EMDP (10 and 100 microM) followed by incubation with testosterone at a V(max) concentration (50 microM). Racemic methadone, EDDP, and EMDP did not act as competitive inhibitors of CYP19. Preincubation of methadone, EDDP, or EMDP with CYP19 resulted in time- and concentration-dependent inhibition, indicating a mechanism-based reaction that destroys CYP19 activity. The K(I) and k(inact) values for racemic methadone were calculated to be 40.6 +/- 2.8 microM and 0.061 +/- 0.001 min(-1), respectively. No stereoselectivity was observed. Methadone is metabolized by CYP19 and may act as a potent inhibitor of CYP19 in vivo. These findings may contribute to variability in methadone clearance, to drug-drug interactions, and to side effects observed in individual patients.

  19. Pharmacophore modeling and in silico screening for new P450 19 (aromatase) inhibitors.

    PubMed

    Schuster, Daniela; Laggner, Christian; Steindl, Theodora M; Palusczak, Anja; Hartmann, Rolf W; Langer, Thierry

    2006-01-01

    Cytochrome P450 19 (P450 19, aromatase) constitutes a successful target for the treatment of breast cancer. This study analyzes chemical features common to P450 19 inhibitors to develop ligand-based, selective pharmacophore models for this enzyme. The HipHop and HypoRefine algorithms implemented in the Catalyst software package were employed to create both common feature and quantitative models. The common feature model for P450 19 includes two ring aromatic features in its core and two hydrogen bond acceptors at the ends. The models were used as database search queries to identify active compounds from the NCI database.

  20. Erythema multiforme after radiotherapy with aromatase inhibitor administration in breast-conservation treatment for breast cancer.

    PubMed

    Nakatani, Kimiko; Matsumoto, Masaaki; Ue, Hironobu; Nishioka, Akihito; Tanaka, Yousuke; Kodama, Hajime; Sasaguri, Shiro; Ogawa, Yasuhiro

    2008-01-01

    Generalized eruptions associated with radiotherapy such as erythema multiforme (EM), Steven-Johnson syndrome and toxic epidermal necrolysis are uncommon reactions. A few cases of generalized eruptions during and after radiotherapy have been reported with the use of anticonvulsants and anticancer drugs. However, no reports have described mucocutaneous reactions associated with radiotherapy and concurrent use of anastrozole, an aromatase inhibitor. This report describes EM occurring after radiotherapy performed during breast-conserving treatment for breast cancer in a patient who was taking oral anastrozole.

  1. Synthesis and PET studies of [11C-cyano]letrozole (Femara), an aromatase inhibitor drug

    SciTech Connect

    kil K. E.; Biegon A.; Kil, K.-E.; Biegon, A.; Ding, Y.-S.; Fischer, A.; Ferrieri, R.A.; Kim, S.-W.; Pareto, D.; Schueller, M.J.; Fowler, J.S.

    2008-11-10

    Aromatase, a member of the cytochrome P450 family, converts androgens such as androstenedione and testosterone to estrone and estradiol respectively. Letrozole (1-[bis-(4-cyanophenyl)methyl]-1H-1,2,4-triazole, Femara{reg_sign}) is a high affinity aromatase inhibitor (K{sub i}=11.5 nM) which has FDA approval for breast cancer treatment. Here we report the synthesis of carbon-11 labeled letrozole and its assessment as a radiotracer for brain aromatase in the baboon. Letrozole and its precursor (4-[(4-bromophenyl)-1H-1,2,4-triazol-1-ylmethyl]benzonitrile, 3) were prepared in two-step syntheses from 4-cyanobenzyl bromide and 4-bromobenzyl bromide, respectively. The [{sup 11}C]cyano group was introduced via the tetrakis(triphenylphosphine)palladium(0) catalyzed coupling of [{sup 11}C]cyanide with the bromo-precursor (3). PET studies in the baboon brain were carried out to assess regional distribution and kinetics, reproducibility of repeated measures and saturability. The free fraction of letrozole in the plasma, log D, and the [{sup 11}C-cyano]letrozole fraction in the arterial plasma were also measured. [{sup 11}C-cyano]Letrozole was synthesized in 60 min with a radiochemical yield of 79-80%, with a radiochemical purity greater than 98% and a specific activity of 4.16 {+-} 2.21 Ci/{micro}mol at the end of bombardment (n=4). PET studies in the baboon revealed initial rapid and high uptake and initial rapid clearance followed by slow clearance of carbon-11 from the brain with no difference between brain regions. The brain kinetics was not affected by co-injection of unlabeled letrozole (0.1 mg/kg). The free fraction of letrozole in plasma was 48.9% and log D was 1.84. [{sup 11}C-cyano]Letrozole is readily synthesized via a palladium catalyzed coupling reaction with [{sup 11}C]cyanide. Although it is unsuitable as a PET radiotracer for brain aromatase as revealed by the absence of regional specificity and saturability in brain regions, such as amygdala, which are known

  2. Three-dimensional quantitative structure-activity relationships of steroid aromatase inhibitors

    NASA Astrophysics Data System (ADS)

    Oprea, Tudor I.; García, Angel E.

    1996-06-01

    Inhibition of aromatase, a cytochrome P450 that converts androgens to estrogens, is relevant in the therapeutic control of breast cancer. We investigate this inhibition using a three-dimensional quantitative structure-activity relationship (3D QSAR) method known as Comparative Molecular Field Analysis, CoMFA [Cramer III, R.D. et al., J. Am. Chem. Soc., 110 (1988) 5959]. We analyzed the data for 50 steroid inhibitors [Numazawa, M. et al., J. Med. Chem., 37 (1994) 2198, and references cited therein] assayed against androstenedione on human placental microsomes. An initial CoMFA resulted in a three-component model for log(1/Ki), with an explained variance r2 of 0.885, and a cross-validated q2 of 0.673. Chemometric studies were performed using GOLPE [Baroni, M. et al., Quant. Struct.-Act. Relatsh., 12 (1993) 9]. The CoMFA/GOLPE model is discussed in terms of robustness, predictivity, explanatory power and simplicity. After randomized exclusion of 25 or 10 compounds (repeated 25 times), the q2 for one component was 0.62 and 0.61, respectively, while r2 was 0.674. We demonstrate that the predictive r2 based on the mean activity (Ym) of the training set is misleading, while the test set Ym-based predictive r2 index gives a more accurate estimate of external predictivity. Using CoMFA, the observed differences in aromatase inhibition among C6-substituted steroids are rationalized at the atomic level. The CoMFA fields are consistent with known, potent inhibitors of aromatase, not included in the model. When positioned in the same alignment, these compounds have distinct features that overlap with the steric and electrostatic fields obtained in the CoMFA model. The presence of two hydrophobic binding pockets near the aromatase active site is discussed: a steric bulk tolerant one, common for C4, C6-alpha and C7-alpha substitutents, and a smaller one at the C6-beta region.

  3. Aromatase inhibitors in obese breast cancer patients are not associated with increased plasma estradiol levels.

    PubMed

    Diorio, Caroline; Lemieux, Julie; Provencher, Louise; Hogue, Jean-Charles; Vachon, Eric

    2012-11-01

    Obesity, in postmenopausal women, has been associated to a higher breast cancer incidence and worst prognosis. Some studies suggested a decrease in aromatase inhibitors (AI) efficacy in obese postmenopausal breast cancer patients, although estradiol levels were not measured. The purpose of the present study was to verify if estradiol levels are measurable in postmenopausal women under AI. If achievable, the goal is to compare the estradiol levels in lean versus obese postmenopausal women under AI treatment for non-metastatic breast cancer. Postmenopausal women were recruited in accordance to one of these four groups: lean [body mass index (BMI) of 18-25 kg/m(2)] under AI (n = 30), obese (BMI ≥ 30 kg/m(2)) under AI (n = 30), lean AI-naïve (n = 10), and obese AI-naïve (n = 10). Lean and obese women were matched according to their age. Estradiol levels were measured in plasma using an ELISA. The Wilcoxon signed-rank test was used to assess the significance of the differences between the groups. Estradiol levels in postmenopausal women under AI varied from 0 to 94.65 pg/ml with a median value of 0.98 pg/ml. Obese AI-naïve women had higher estradiol levels than lean AI-naïve women (p = 0.03). There was no difference in estradiol levels between lean and obese women under AI (p = 0.76). Despite very low plasma levels, it is possible to measure the estradiol levels in postmenopausal women under AI treatment. Our results suggest that the known impact of obesity on recurrence risk in women under AI treatment may not be due to incomplete aromatase inhibition. Further works are needed to examine closely the aromatase-independent pathways that are linking obesity to breast cancer risk and recurrence.

  4. The case for aromatase inhibitors use in oncofertility patients. Should aromatase inhibitors be combined with gonadotropin treatment in breast cancer patients undergoing ovarian stimulation for fertility preservation prior to chemotherapy? A debate.

    PubMed

    Fatum, Muhammad; McVeigh, Enda; Child, Tim

    2013-12-01

    Breast cancer is one of the hormone-dependent cancers that may be adversely affected by elevated oestrogen or progesterone concentrations, particularly the endocrine active (hormone receptor positive) breast cancers. Treatment for breast cancer patients aimed at fertility preservation, includes ovarian hyperstimulation, the harvest of oocytes, and subsequent cryopreservation of oocytes or embryos. Classically, gonadotrophins have been used effectively for ovulation induction, a treatment often accompanied by high blood oestrogen concentrations produced by the hyperstimulated granulosa cells. Despite the uncertainty which surrounds this issue and the lack of clear-cut clinical evidence, it is still of major concern that these ensuing high hormone levels might be associated with a high risk of recurrence of the cancer. A growing number of clinical studies have strongly suggested the benefits of using aromatase inhibitors in infertility treatment, both as single agents or as adjuncts to FSH-containing ovulation induction regimes in reproductive medicine. Combining gonadotrophins with aromatase inhibitors would augment the stimulation effect, with a reduced increase in serum concentrations of estradiol. We propose to open a debate over the use of aromatase inhibitors in combination with FSH in ovulation induction treatment of breast cancer oncofertility patients. As the safety of aromatase inhibitors such as letrozole has recently been demonstrated in several studies, and there is growing concern over the possible detrimental effects of high estradiol levels on breast cancer cells (at least in mouse models), the co-administration of letrozole in these patients would reduce both the high supraphysiologic serum levels of estradiol and the intratumoral in situ production of oestrogen. However, since it is unlikely that a well-founded evidence-based justification of this treatment will be formulated in the near future, based on well-designed prospective randomised

  5. EVALUATION OF THE AROMATASE INHIBITOR FADROZOLE IN A SHORT-TERM REPRODUCTION ASSAY WITH THE FATHEAD MINNOW (PIMEPHALES PROMELAS)

    EPA Science Inventory

    Cytochrome P450 aromatase is a key enzyme in vertebrate steroidogenesis, catalyzing the conversion of C19 androgens to C18 estrogens such a B-estradiol (E2). The objective of this study was to assess effects of the CYP inhibitor fadrozole on fathead minnow reproductive endocrinol...

  6. Effects of a short-term exposure to the aromatase inhibitor fadrozole on steroid production and gene expression in the ovary of female fathead minnows (Pimephales promelas)

    EPA Science Inventory

    Cytochrome P450 aromatase is a steriodogenic enzyme that converts C19 androgens to C18 estrogens and is critical for normal reproduction in females. Fadrozole is a well-studied aromatase inhibitor that has been shown to suppress estrogen production in the ovaries of fish. Howev...

  7. Management of sexual dysfunction in postmenopausal breast cancer patients taking adjuvant aromatase inhibitor therapy

    PubMed Central

    Derzko, C.; Elliott, S.; Lam, W.

    2007-01-01

    Treatment with aromatase inhibitors for postmenopausal women with breast cancer has been shown to reduce or obviate invasive procedures such as hysteroscopy or curettage associated with tamoxifen-induced endometrial abnormalities. The side effect of upfront aromatase inhibitors, diminished estrogen synthesis, is similar to that seen with the natural events of aging. The consequences often include vasomotor symptoms (hot flushes) and vaginal dryness and atrophy, which in turn may result in cystitis and vaginitis. Not surprisingly, painful intercourse (dyspareunia) and loss of sexual interest (decreased libido) frequently occur as well. Various interventions, both non-hormonal and hormonal, are currently available to manage these problems. The purpose of the present review is to provide the practitioner with a wide array of management options to assist in treating the sexual consequences of aromatase inhibitors. The suggestions in this review are based on recent literature and on the recommendations set forth both by the North American Menopause Association and in the clinical practice guidelines of the Society of Gynaecologists and Obstetricians of Canada. The complexity of female sexual dysfunction necessitates a biopsychosocial approach to assessment and management alike, with interventions ranging from education and lifestyle changes to sexual counselling, pelvic floor therapies, sexual aids, medications, and dietary supplements—all of which have been reported to have a variable, but often successful, effect on symptom amelioration. Although the use of specific hormone replacement—most commonly local estrogen, and less commonly, systemic estrogen with or without an androgen, progesterone, or the additional of an androgen in an estrogenized woman (or a combination)—may be highly effective, the concern remains that in patients with estrogen-dependent breast cancer, including those receiving anti-estrogenic adjuvant therapies, the use of these hormones may be

  8. Management of sexual dysfunction in postmenopausal breast cancer patients taking adjuvant aromatase inhibitor therapy.

    PubMed

    Derzko, C; Elliott, S; Lam, W

    2007-12-01

    Treatment with aromatase inhibitors for postmenopausal women with breast cancer has been shown to reduce or obviate invasive procedures such as hysteroscopy or curettage associated with tamoxifen-induced endometrial abnormalities. The side effect of upfront aromatase inhibitors, diminished estrogen synthesis, is similar to that seen with the natural events of aging. The consequences often include vasomotor symptoms (hot flushes) and vaginal dryness and atrophy, which in turn may result in cystitis and vaginitis. Not surprisingly, painful intercourse (dyspareunia) and loss of sexual interest (decreased libido) frequently occur as well. Various interventions, both non-hormonal and hormonal, are currently available to manage these problems. The purpose of the present review is to provide the practitioner with a wide array of management options to assist in treating the sexual consequences of aromatase inhibitors. The suggestions in this review are based on recent literature and on the recommendations set forth both by the North American Menopause Association and in the clinical practice guidelines of the Society of Gynaecologists and Obstetricians of Canada. The complexity of female sexual dysfunction necessitates a biopsychosocial approach to assessment and management alike, with interventions ranging from education and lifestyle changes to sexual counselling, pelvic floor therapies, sexual aids, medications, and dietary supplements-all of which have been reported to have a variable, but often successful, effect on symptom amelioration. Although the use of specific hormone replacement-most commonly local estrogen, and less commonly, systemic estrogen with or without an androgen, progesterone, or the additional of an androgen in an estrogenized woman (or a combination)-may be highly effective, the concern remains that in patients with estrogen-dependent breast cancer, including those receiving anti-estrogenic adjuvant therapies, the use of these hormones may be

  9. Review of hormone-based treatments in postmenopausal patients with advanced breast cancer focusing on aromatase inhibitors and fulvestrant

    PubMed Central

    Kümler, Iben; Knoop, Ann S; Jessing, Christina A R; Ejlertsen, Bent; Nielsen, Dorte L

    2016-01-01

    Background Endocrine therapy constitutes a central modality in the treatment of oestrogen receptor (ER)-positive advanced breast cancer. Purpose To evaluate the evidence for endocrine treatment in postmenopausal patients with advanced breast cancer focusing on the aromatase inhibitors, letrozole, anastrozole, exemestane and fulvestrant. Methods A review was carried out using PubMed. Randomised phase II and III trials reporting on ≥100 patients were included. Results 35 trials met the inclusion criteria. If not used in the adjuvant setting, a non-steroid aromatase inhibitor was the optimal first-line option. In general, the efficacy of the different aromatase inhibitors and fulvestrant was similar in tamoxifen-refractory patients. A randomised phase II trial of palbociclib plus letrozole versus letrozole alone showed significantly increased progression-free survival (PFS) when compared with endocrine therapy alone in the first-line setting (20.2 vs 10.2 months). Furthermore, the addition of everolimus to exemestane in the Breast Cancer Trials of OraL EveROlimus-2 (BOLERO-2) study resulted in an extension of median PFS by 4.5 months after recurrence/progression on a non-steroid aromatase inhibitor. However, overall survival was not significantly increased. Conclusion Conventional treatment with an aromatase inhibitor or fulvestrant may be an adequate treatment option for most patients with hormone receptor-positive advanced breast cancer. Mammalian target of rapamycin (mTOR) inhibition and cyclin-dependent kinase 4/6 (CDK4/6) inhibition might represent substantial advances for selected patients in some specific settings. However, there is an urgent need for prospective biomarker-driven trials to identify patients for whom these treatments are cost-effective. PMID:27843622

  10. Multiple Structural and Functional Abnormalities in the P450 Aromatase Expressing Transgenic Male Mice Are Ameliorated by a P450 Aromatase Inhibitor

    PubMed Central

    Li, Xiangdong; Strauss, Leena; Mäkelä, Sari; Streng, Tomi; Huhtaniemi, Ilpo; Santti, Risto; Poutanen, Matti

    2004-01-01

    The present study was undertaken to analyze the effect of a P450 aromatase inhibitor (finrozole) on 4-month-old transgenic mice expressing human P450 aromatase (P450arom) under the human ubiquitin C promoter (AROM+). AROM+ mice present several dysfunctions, such as adrenal and pituitary hyperplasia, cryptorchidism, Leydig cell hypertrophy and hyperplasia, and gynecomastia. The present study demonstrates that these abnormalities were efficiently treated by administration of a P450arom inhibitor, finrozole. The treatment normalized the reduced intratesticular and serum testosterone levels, while those of estradiol were decreased. The body weight and several affected organ weights were normalized with the treatment. Histological analysis revealed that both the pituitary and adrenal hyperplasia were diminished. Furthermore, the cryptorchid testes present in the untreated AROM+ males descended to scrotum, 4 to 15 days after inhibitor treatment. In addition, the disrupted spermatogenesis was recovered and qualitatively complete spermatogenesis appeared with the inhibitor treatment. This was associated with normalized structure of the interstitial tissue, as analyzed by immunohistochemical staining for Leydig cells and macrophages. One of the features was that the Leydig cell hypertrophy was markedly diminished in the treated mice. AROM+ mice also present with severe gynecomastia, while the development and differentiation of the mammary gland in AROM+ males was markedly diminished with the inhibitor treatment. Interestingly, the mammary gland involution was associated with the induction of androgen receptor in the epithelial cells, while estrogen receptors were still detectable in the epithelium. The data show that AROM+ mouse model is a novel tool to further analyze the use of P450arom inhibitors in the treatment of the dysfunctions in males associated with misbalanced estrogen to androgen ratio, such as pituitary adenoma, testicular dysfunction, and gynecomastia. PMID

  11. Gas chromatography-mass spectrometric study of 19-oxygenation of the aromatase inhibitor 19-methylandrostenedione with human placental microsomes.

    PubMed

    Numazawa, Mitsuteru; Nagaoka, Masao; Handa, Wakako; Yamada, Akane

    2006-06-01

    To gain insight into the catalytic function of aromatase, we studied 19-oxygenation of 19-methyl-substituted derivative of the natural substrate androstenedione (AD), compound 1, with human placental aromatase by use of gas chromatography-mass spectrometry (GC-MS). Incubation of the 19-methyl derivative 1 with human placental microsomes in the presence of NADPH under an aerobic condition did not yield a detectable amount of [19S]19-hydroxy product 2 or its [19R]-isomer 3 when the product was analyzed as the bis-methoxime-trimethylsilyl (TMS) derivative by GC-MS; moreover, the production of estrogen was not detected as the bis-TMS derivative of estradiol (detection limit: about 3 ng and 10 pg per injection for the 19-ol and estradiol, respectively). The results reveal that the 19-methyl steroid 1 does not serve as a substrate of aromatase, although it does serve as a powerful inhibitor of the enzyme.

  12. Structure-activity relationships of 2alpha-substituted androstenedione analogs as aromatase inhibitors and their aromatization reactions.

    PubMed

    Numazawa, Mitsuteru; Handa, Wakako; Hasegawa, Chie; Takahashi, Madoka

    2005-12-01

    Aromatase catalyzes the conversion of androstenedione (1a, AD) to estrone through three sequential oxygenations of the 19-methyl group. To gain insight into the spatial nature of the AD binding (active) site of aromatase in relation to the catalytic function of the enzyme, we tested for the ability of 2alpha-substituted (halogeno, alkyl, hydroxy, and alkoxy) ADs (1b-1i) to inhibit aromatase in human placental microsomes as well as their ability to serve as a substrate for the enzyme. All of the steroids inhibited the enzyme in a competitive manner with the apparent K(i)'s ranging from 45 to 1150 nM. 2alpha-Halogeno (F, Cl, and Br) and 2alpha-alkyl (CH3 and CH2CH3) steroids 1b-1f were powerful to good inhibitors (Ki=45-171 nM) whereas steroids 1g-1i, having an oxygen function (hydroxy or alkoxy) at C-2alpha, were poor inhibitors (Ki=670-1150 nM). Aromatization of some of the steroids with placental microsomes was analyzed by gas chromatography-mass spectrometry, indicating that the aromatization rate of the bromide 1d was about two-fold that of the natural substrate AD and that of 2alpha-methoxide 1h was similar to that of AD. Kinetic analysis of the aromatization of androgens revealed that a good substrate was not essentially a good inhibitor for aromatase.

  13. Induction of Female-to-Male Sex Change in Adult Zebrafish by Aromatase Inhibitor Treatment

    NASA Astrophysics Data System (ADS)

    Takatsu, Kanae; Miyaoku, Kaori; Roy, Shimi Rani; Murono, Yuki; Sago, Tomohiro; Itagaki, Hideyuki; Nakamura, Masaru; Tokumoto, Toshinobu

    2013-12-01

    This study investigated whether undifferentiated germ and/or somatic stem cells remain in the differentiated ovary of a species that does not undergo sex changes under natural conditions and retain their sexual plasticity. The effect of aromatase inhibitor (AI)-treatment on sexually mature female zebrafish was examined. A 5-month AI treatment caused retraction of the ovaries after which testes-like organs appeared, and cyst structures filled with spermatozoa-like cells were observed in sections of these tissues. Electron microscopic observations revealed that these cells appeared as large sperm heads without tails. Sperm formation was re-examined after changing the diet to an AI-free food. A large number of normal sperm were obtained after eight weeks, and no formation of ovarian tissue was observed. Artificial fertilization using sperm from the sex-changed females was successful. These results demonstrated that sex plasticity remains in the mature ovaries of this species.

  14. Induction of female-to-male sex change in adult zebrafish by aromatase inhibitor treatment.

    PubMed

    Takatsu, Kanae; Miyaoku, Kaori; Roy, Shimi Rani; Murono, Yuki; Sago, Tomohiro; Itagaki, Hideyuki; Nakamura, Masaru; Tokumoto, Toshinobu

    2013-12-02

    This study investigated whether undifferentiated germ and/or somatic stem cells remain in the differentiated ovary of a species that does not undergo sex changes under natural conditions and retain their sexual plasticity. The effect of aromatase inhibitor (AI)-treatment on sexually mature female zebrafish was examined. A 5-month AI treatment caused retraction of the ovaries after which testes-like organs appeared, and cyst structures filled with spermatozoa-like cells were observed in sections of these tissues. Electron microscopic observations revealed that these cells appeared as large sperm heads without tails. Sperm formation was re-examined after changing the diet to an AI-free food. A large number of normal sperm were obtained after eight weeks, and no formation of ovarian tissue was observed. Artificial fertilization using sperm from the sex-changed females was successful. These results demonstrated that sex plasticity remains in the mature ovaries of this species.

  15. Hybrid flavan-chalcones, aromatase and lipoxygenase inhibitors, from Desmos cochinchinensis.

    PubMed

    Bajgai, Santi Prasad; Prachyawarakorn, Vilailak; Mahidol, Chulabhorn; Ruchirawat, Somsak; Kittakoop, Prasat

    2011-11-01

    Hybrid flavan-chalcones, desmosflavans A (1) and B (2), together with three known compounds, cardamonin (3), pinocembrin (4) and chrysin (5), were isolated from leaves of Desmos cochinchinensis. Cardamonin (3) and chrysin (5) exhibited potent antioxidant activity with 15.0 and 12.2 ORAC units. Desmosflavans A (1) and B (2), pinocembrin (4), and chrysin (5) were found to be inhibitors of aromatase with respective IC50 values of 1.8, 3.3, 0.9, and 0.8 μM. Desmosflavan A (1) inhibited lipoxygenase with the IC50 value of 4.4 μM. Desmosflavan A (1) exhibited cytotoxic activity with IC50 values of 0.29-3.75 μg/mL, while desmosflavan B (2) showed IC50 values of 1.71-27.0 μg/mL.

  16. Growth Hormone With Aromatase Inhibitor May Improve Height in CYP11B1 Congenital Adrenal Hyperplasia.

    PubMed

    Hawton, Katherine; Walton-Betancourth, Sandra; Rumsby, Gill; Raine, Joseph; Dattani, Mehul

    2017-02-01

    With an estimated prevalence of 1 in 100 000 births, 11β-hydroxylase deficiency is the second most common form of congenital adrenal hyperplasia (CAH) and is caused by mutations in CYP11B1 Clinical features include virilization, early gonadotropin-independent precocious puberty, hypertension, and reduced stature. The current mainstay of management is with glucocorticoids to replace deficient steroids and to minimize adrenal sex hormone overproduction, thus preventing virilization and optimizing growth. We report a patient with CAH who had been suboptimally treated and presented to us at 6 years of age with precocious puberty, hypertension, tall stature, advanced bone age, and a predicted final height of 150 cm. Hormonal profiles and genetic analysis confirmed a diagnosis of 11β-hydroxylase deficiency. In addition to glucocorticoid replacement, the patient was commenced on growth hormone and a third-generation aromatase inhibitor, anastrozole, in an attempt to optimize his growth. After the initiation of this treatment, the patient's growth rate improved significantly and bone age advancement slowed. The patient reached a final height of 177.5 cm (0.81 SD score), 11.5 cm above his mid-parental height. This patient is only the second reported case of the use of an aromatase inhibitor in combination with growth hormone to optimize height in 11β-hydroxylase-deficient CAH. This novel treatment proved to be highly efficacious, with no adverse effects. It may therefore provide a promising option to promote growth in exceptional circumstances in individuals with 11β-hydroxylase deficiency presenting late with advanced skeletal maturation and consequent short stature.

  17. Potential of aromatase inhibitors for ovulation and superovulation induction in infertile women.

    PubMed

    Mitwally, Mohamed F M; Casper, Robert F

    2006-01-01

    For almost half a century, the first-line treatment for ovulation induction in cases of anovulation, unexplained infertility, or mild male factor has been clomifene (clomiphene citrate). Clomifene is an effective and safely used oral agent, but is known to have relatively common antiestrogenic endometrial and cervical mucous adverse effects that could prevent pregnancy in the face of successful ovulation. In addition, there is a significant risk of multiple pregnancies with clomifene compared with natural cycles. These drawbacks are mainly a result of the extended antiestrogenic effect of clomifene as a result of its accumulation in the body (clomifene isomers have a half-life of several days up to few weeks). Because of these problems, we proposed the concept of aromatase inhibition as a new method of ovulation induction that could avoid many of the adverse effects of clomifene. Over the last few years several published studies, both controlled and noncontrolled, compared clomifene and treatment with aromatase inhibitors (AIs), either alone or in combination with gonadotropins, for ovulation induction or augmentation. These studies found AIs as effective as clomifene in inducing ovulation, with the major advantage of absence of any antiestrogenic adverse effects. Several other major advantages of AIs include the lower serum estrogen production per developing follicle resulting in more physiological estrogen levels around the time of ovulation and good pregnancy rates with a lower incidence of multiple pregnancy than with clomifene. When combined with gonadotropins for assisted reproductive technologies, AIs reduce the dose of gonadotropins required for optimal follicle recruitment and improve the response to gonadotropin stimulation in poor responders. Such preliminary evidence suggests that AIs may replace clomifene in the future because of similar efficacy with a reduced adverse-effect profile. However, we believe that definitive studies in the form of

  18. A retrospective study on screening and management of osteoporosis in breast cancer women treated with aromatase inhibitors in Libya.

    PubMed

    Abushwereb, Hanan Saaddedin; Elhabash, M; Elhamshari, S; Eshaefi, H

    2016-01-01

    Negative health effects of aromatase inhibitors (AI) treatments on bones such as osteoporosis are evidenced. This impact of the aromatase inhibitors on bone. This study aimed to improve the medical assistance given to patients under AI treatment to minimize secondary osteoporosis. Fifty Libyan postmenopausal women treated with AI to fight breast cancer were selected from attendants Tripoli Medical Center (TMC), Oncology Department during year 2014. A closed questionnaire was requested from each women including data about age, age at AI therapy, and types of AI, age at bone densitometry measurement, onset and symptoms of osteoporosis, treatment of osteoporosis and measurement of vitamin D and calcium supplement given. The study revealed a poor consideration given to apply the recommendation in cases suffering osteoporosis events. Our results suggest an active implementation of the guidelines concerning the high corporation levels that should be done between oncologist, specialist in osteoporosis, and patients to offer reliable diagnostic and post-therapy follow up.

  19. A retrospective study on screening and management of osteoporosis in breast cancer women treated with aromatase inhibitors in Libya

    PubMed Central

    Abushwereb, Hanan Saaddedin; Elhabash, M.; Elhamshari, S.; Eshaefi, H.

    2016-01-01

    Summary Negative health effects of aromatase inhibitors (AI) treatments on bones such as osteoporosis are evidenced. This impact of the aromatase inhibitors on bone. This study aimed to improve the medical assistance given to patients under AI treatment to minimize secondary osteoporosis. Fifty Libyan postmenopausal women treated with AI to fight breast cancer were selected from attendants Tripoli Medical Center (TMC), Oncology Department during year 2014. A closed questionnaire was requested from each women including data about age, age at AI therapy, and types of AI, age at bone densitometry measurement, onset and symptoms of osteoporosis, treatment of osteoporosis and measurement of vitamin D and calcium supplement given. The study revealed a poor consideration given to apply the recommendation in cases suffering osteoporosis events. Our results suggest an active implementation of the guidelines concerning the high corporation levels that should be done between oncologist, specialist in osteoporosis, and patients to offer reliable diagnostic and post-therapy follow up. PMID:28228779

  20. The role of aromatase inhibitors in ameliorating deleterious effects of ovarian stimulation on outcome of infertility treatment

    PubMed Central

    Mitwally, Mohamed FM; Casper, Robert F; Diamond, Michael P

    2005-01-01

    Clinical utilization of ovulation stimulation to facilitate the ability of a couple to conceive has not only provided a valuable therapeutic approach, but has also yielded extensive information on the physiology of ovarian follicular recruitment, endometrial receptivity and early embryo competency. One of the consequences of the use of fertility enhancing agents for ovarian stimulation has been the creation of a hyperestrogenic state, which may influence each of these parameters. Use of aromatase inhibitors reduces hyperestrogenism inevitably attained during ovarian stimulation. In addition, the adjunct use of aromatase inhibitors during ovarian stimulation reduces amount of gonadotropins required for optimum stimulation. The unique approach of reducing hyperestrogenism, as well as lowering amount of gonadotropins without affecting the number of mature ovarian follicles is an exciting strategy that could result in improvement in the treatment outcome by ameliorating the deleterious effects of the ovarian stimulation on follicular development, endometrial receptivity, as well as oocyte and embryo quality. PMID:16202169

  1. Comparison of Subjective and Objective Hot Flash Measures Over Time Among Breast Cancer Survivors Initiating Aromatase Inhibitor Therapy

    PubMed Central

    Otte, Julie L.; Flockhart, David; Storniolo, Anna Maria; Schneider, Bryan; Azzouz, Faouzi; Lemler, Suzanne; Jeter, Stacie; Carpenter, Janet S.; Hayes, Daniel; Stearns, Vered; Henry, N. Lynn; Nguyen, Anne; Hayden, Jill; Wright, Laurie

    2009-01-01

    Objective Hot flashes are valuable indicators of physiologic condition and drug effect; however, subjective and objective measures do not always agree. No study has examined both subjective and objective hot flashes in women prescribed aromatase inhibitors. The study (1) compared subjective and objective hot flash measures, (2) examined changes in subjective and objective hot flashes over time, and (3) evaluated predictors of change in hot flashes in aromatase inhibitor-treated women. Design Subjects (n=135) were enrolled in a randomized clinical trial comparing exemestane and letrozole for the treatment of breast cancer. Hot flashes were assessed prior to starting drug and 1, 3, and 6 months later. Subjects wore a sternal skin conductance monitor for ≥ 24 hours at each time point. With each perceived hot flash, women pressed an event button and rated intensity and bother in a paper diary. Results Subjects were a mean age of 60 years and mainly Caucasian (92%). Across time points, monitor hot flashes were (1) significantly more frequent than diary and/or event button flashes (p<.05) and (2) moderately correlated with subjective measures (.35< r <.56). Monitor hot flashes did not significantly change over time with aromatase inhibitor therapy, whereas both diary and event button frequency significantly varied but in dissimilar patterns (51% non-linear). No consistent predictors of hot flashes across measures or time points were identified. Conclusions Findings indicated dissimilarities between subjective and objective measures of hot flashes. Despite statistical significance, there was little clinically meaningful change in hot flashes after initiating aromatase inhibitor therapy. PMID:19455068

  2. Novel highly potent and selective nonsteroidal aromatase inhibitors: synthesis, biological evaluation and structure-activity relationships investigation.

    PubMed

    Gobbi, Silvia; Zimmer, Christina; Belluti, Federica; Rampa, Angela; Hartmann, Rolf W; Recanatini, Maurizio; Bisi, Alessandra

    2010-07-22

    In further pursuing our search for potent and selective aromatase inhibitors, a new series of molecules was designed and synthesized, exploring possible structural modifications of a previously identified xanthone scaffold. Among them, highly potent compounds, with inhibitory activity in the low nanomolar range, were found. In particular, substitution of the heterocyclic oxygen atom in the xanthone core by a sulfur atom and/or increase in structure flexibility seemed to be favorable for the interaction with the enzyme.

  3. Aromatase inhibition 2013: clinical state of the art and questions that remain to be solved

    PubMed Central

    Lønning, Per Eystein; Eikesdal, Hans Petter

    2013-01-01

    Following their successful implementation for the treatment of metastatic breast cancer, the ‘third-generation’ aromatase inhibitors (anastrozole, letrozole, and exemestane) have now become standard adjuvant endocrine treatment for postmenopausal estrogen receptor-positive breast cancers. These drugs are characterized by potent aromatase inhibition, causing >98% inhibition of estrogen synthesis in vivo. A recent meta-analysis found no difference in anti-tumor efficacy between these three compounds. As of today, aromatase inhibitor monotherapy and sequential treatment using tamoxifen followed by an aromatase inhibitor for a total of 5 years are considered equipotent treatment options. However, current trials are addressing the potential benefit of extending treatment duration beyond 5 years. Regarding side effects, aromatase inhibitors are not found associated with enhanced risk of cardiovascular disease, and enhanced bone loss is prevented by adding bisphosphonates in concert for those at danger of developing osteoporosis. However, arthralgia and carpal tunnel syndrome preclude drug administration among a few patients. While recent findings have questioned the use of aromatase inhibitors among overweight and, in particular, obese patients, this problem seems to focus on premenopausal patients treated with an aromatase inhibitor and an LH-RH analog in concert, questioning the efficacy of LH-RH analogs rather than aromatase inhibitors among overweight patients. Finally, recent findings revealing a benefit from adding the mTOR inhibitor everolimus to endocrine treatment indicate targeted therapy against defined growth factor pathways to be a way forward, by reversing acquired resistance to endocrine therapy. PMID:23625614

  4. Effects of a purported aromatase and 5α-reductase inhibitor on hormone profiles in college-age men.

    PubMed

    Wilborn, Colin; Taylor, Lem; Poole, Chris; Foster, Cliffa; Willoughby, Darryn; Kreider, Richard

    2010-12-01

    The purpose of this study was to determine the effects of an alleged aromatase and 5-α reductase inhibitor (AI) on strength, body composition, and hormonal profiles in resistance-trained men. Thirty resistance-trained men were randomly assigned in a double-blind manner to ingest 500 mg of either a placebo (PL) or AI once per day for 8 wk. Participants participated in a 4-d/wk resistance-training program for 8 wk. At Weeks 0, 4, and 8, body composition, 1-repetition-maximum (1RM) bench press and leg press, muscle endurance, anaerobic power, and hormonal profiles were assessed. Statistical analyses used a 2-way ANOVA with repeated measures for all criterion variables (p ≤ .05). Significant Group × Time interaction effects occurred over the 8-wk period for percent body fat (AI: -1.77% ± 1.52%, PL: -0.55% ± 1.72%; p = .048), total testosterone (AI: 0.97 ± 2.67 ng/ml, PL: -2.10 ± 3.75 ng/ml; p = .018), and bioavailable testosterone (AI: 1.32 ± 3.45 ng/ml, PL: -1.69 ± 3.94 ng/ml; p = .049). Significant main effects for time (p ≤ .05) were noted for bench- and leg-press 1RM, lean body mass, and estradiol. No significant changes were detected among groups for Wingate peak or mean power, total body weight, dihydrotestosterone, hemodynamic variables, or clinical safety data (p > .05). The authors concluded that 500 mg of dailyAI supplementation significantly affected percent body fat, total testosterone, and bioavailable testosterone compared with a placebo in a double-blind fashion.

  5. Sensitivity of New Zealand mudsnail Potamopyrgus antipodarum (Gray) to a specific aromatase inhibitor.

    PubMed

    Gust, M; Garric, J; Giamberini, L; Mons, R; Abbaci, K; Garnier, F; Buronfosse, T

    2010-03-01

    The freshwater prosobranch Potamopyrgus antipodarum (Molluska, Hydrobiidea, Smith 1889) has been proposed as a suitable species to assess the impact of endocrine disrupting compounds (EDC) in aquatic ecosystems. Steroid hormone biosynthesis pathway is potentially an important target for EDC, and vertebrate-like sex steroids seem to play a functional role in the control of mollusk reproduction. To assess the response and the sensitivity of P. antipodarum to disrupters of the steroid hormone biosynthesis pathway, we have experienced the action of a specific vertebrate aromatase inhibitor, fadrozole, acting on 17beta-estradiol synthesis in two separate 28 and 42d exposures. Fadrozole had effects consistent with the expected mechanism of action. A decrease of the reproduction parameters (such as on the number of neonates and number of embryos in the brood pouch) in a dose-dependant manner was observed. The steroids levels were also impaired with the ratio 17beta-estradiol/testosterone decreased by half in exposed snails. This shift of the steroids balance was accompanied by some alteration in the gonads histology and immunohistochemistry in fadrozole-exposed snails. This study highlights the value role of P. antipodarum as a test species for assessing EDC effects in aquatic wildlife.

  6. Endocrinological and clinical evaluation of exemestane, a new steroidal aromatase inhibitor.

    PubMed Central

    Zilembo, N.; Noberasco, C.; Bajetta, E.; Martinetti, A.; Mariani, L.; Orefice, S.; Buzzoni, R.; Di Bartolomeo, M.; Di Leo, A.; Laffranchi, A.

    1995-01-01

    The androstenedione derivative, exemestane (FCE 24304), is a new orally active irreversible aromatase inhibitor. Fifty-six post-menopausal advanced breast cancer patients entered this study to evaluate the activity of four low exemestane doses in reducing oestrogen levels. The drug's tolerability and clinical efficacy were also assessed. Exemestane was orally administered to four consecutive groups at daily doses of 25, 12.5, 5 and 2.5 mg, and the changes in oestrogen, gonadotrophins, sex-hormone binding globulin and dehydroepiandrosterone sulphate levels were evaluated. Drug selectivity was studied by measuring 17-hydroxycorticosteroid urinary levels. After 7 days of treatment, mean oestrone and oestradiol levels had decreased by respectively 64% and 65% (a decrease which was maintained over time); in the 2.5 mg group, oestrone sulphate levels also decreased by 74%. Gonadotrophin levels were significantly higher, whereas no changes in the other serum hormone levels or any interference with adrenal synthesis were detected. Treatment tolerability was satisfactory: nausea and dyspepsia were reported in 16% of patients. The overall objective response rate was 18%. In conclusion, exemestane is effective in reducing oestrogen levels at all of the tested doses and shows interesting clinical activity. PMID:7547212

  7. A Pilot Study of Website Information Regarding Aromatase Inhibitors: Dietary Supplement Interactions

    PubMed Central

    McDermott, Cara L.; Hsieh, Angela A.; Sweet, Erin S.; Tippens, Kimberly M.

    2011-01-01

    Abstract Objectives Patients who have hormone receptor–positive breast cancer and who are taking aromatase inhibitors (AIs) should understand the benefits and risks of concomitant dietary supplement (DS) use. The International Society for Integrative Oncology (SIO) encourages patients to discuss DS use with their health care practitioners. The objective was to conduct a pilot study rating Internet websites from the perspective of health care practitioners for information about AI–DS interactions. Design Five (5) Internet websites suggested by SIO were evaluated using the DISCERN instrument rating tool. The available AI–DS information on these websites was rated by 4 evaluators: 2 naturopathic doctors, 1 oncology pharmacy resident, and a pharmacy student. Results The overall rankings ranged from 1.6 to 3.9, with considerable variability in the type of information available from the websites. The interevaluator rankings of the websites ranged from 0.44 to 0.89. The evaluators consistently found the most reliable, unbiased, and comprehensive information on AI–DS interactions at the Natural Medicines Comprehensive Database and Memorial Sloan-Kettering Cancer Center websites. However, more than one database was needed for provision of optimal patient information on AI–DS interactions. Conclusions In order to effectively advise patients regarding AI–DS interactions, more than one website should be evaluated to assess the potential efficacy and safety of DS in women whose breast cancer is being treated with an AI. PMID:22087614

  8. Vitamin D insufficiency and musculoskeletal symptoms in breast cancer survivors on aromatase inhibitor therapy.

    PubMed

    Waltman, Nancy L; Ott, Carol D; Twiss, Janice J; Gross, Gloria J; Lindsey, Ada M

    2009-01-01

    Breast cancer survivors (BCSs) on aromatase inhibitor (AI) therapy often experience musculoskeletal symptoms (joint pain and stiffness, bone and muscle pain, and muscle weakness), and these musculoskeletal symptoms may be related to low serum levels of vitamin D. The primary purpose of this pilot exploratory study was to determine whether serum levels of 25-hydroxyvitamin D (25[OH]D) concentration were below normal (<30 ng/mL) in 29 BCSs on AI therapy and if musculoskeletal symptoms were related to these low vitamin D levels. The mean (SD) serum 25(OH)D level was 25.62 (4.93) ng/mL; 86% (n = 25) had levels below 30 ng/mL. Patients reported muscle pain in the neck and back, and there was a significant inverse correlation between pain intensity and serum 25(OH)D levels (r = -0.422; P < .05 [2 tailed]). This sample of BCSs taking AIs had below normal levels of serum 25(OH)D despite vitamin D supplements. This is one of the few studies to document a significant relationship between vitamin D levels and muscle pain in BCSs on AI therapy. Findings from this pilot study can be used to inform future studies examining musculoskeletal symptoms in BCSs on AI therapy and relationships with low serum levels of vitamin D.

  9. Postmenopausal women with hormone receptor-positive breast cancer: balancing benefit and toxicity from aromatase inhibitors.

    PubMed

    Ingle, James N

    2013-08-01

    Extensive clinical trial experience is available for aromatase inhibitors (AIs) in postmenopausal women upon which to evaluate the balance of potential benefit and toxicities. A meta-analysis revealed an advantage for AIs over tamoxifen in the monotherapy setting for recurrence but not breast cancer mortality, and an advantage in both of these parameters for switching to an AI after several years of tamoxifen. Importantly, no indication of a deleterious effect of AIs was identified in terms of death without recurrence in these meta-analyses. Regarding serious adverse events (AEs), there are data indicating an increase in cardiovascular AEs and bone fractures but a lower incidence of thromboembolic phenomena and endometrial cancer with AIs vis-à-vis tamoxifen. There does not appear to be a difference in cerebrovascular AEs. Musculoskeletal AEs are the most common clinically important AEs as they are the most common cause of discontinuation of therapy, which can have an adverse effect on outcomes. The balance of benefit and toxicity favors the use of AIs in the adjuvant setting but the absolute benefit from AIs can be decreased in patients with advancing age or increasing comorbidities.

  10. Impact of yoga on functional outcomes in breast cancer survivors with aromatase inhibitor-associated arthralgias.

    PubMed

    Galantino, Mary Lou; Desai, Krupali; Greene, Laurie; Demichele, Angela; Stricker, Carrie Tompkins; Mao, Jun James

    2012-12-01

    Arthralgia affects postmenopausal breast cancer survivors (BCSs) receiving aromatase inhibitors (AIs). This study aims to establish the feasibility of studying the impact of yoga on objective functional outcomes, pain, and health-related quality of life (HR-QOL) for AI-associated arthralgia (AIAA). Postmenopausal women with stage I to III breast cancer who reported AIAA were enrolled in a single-arm pilot trial. A yoga program was provided twice a week for 8 weeks. The Functional Reach (FR) and Sit and Reach (SR) were evaluated as primary outcomes. Pain, as measured by the Brief Pain Inventory (BPI), self-reported Patient Specific Functional Scale (PSFS), and Functional Assessment of Cancer Therapy-Breast (FACT-B) were secondary outcomes. Paired t tests were used for analysis, and 90% provided data for assessment at the end of the intervention. Participants experienced significant improvement in balance, as measured by FR, and flexibility, as measured by SR. The PSFS improved from 4.55 to 7.21, and HR-QOL measured by FACT-B also improved; both P < .05. The score for the Pain Severity subscale of the BPI reduced. No adverse events nor development or worsening of lymphedema was observed. In all, 80% of participants adhered to the home program. Preliminary data suggest that yoga may reduce pain and improve balance and flexibility in BCSs with AIAA. A randomized controlled trial is needed to establish the definitive efficacy of yoga for objective functional improvement in BCSs related to AIAA.

  11. Screening of aromatase inhibitors in traditional Chinese medicines by electrophoretically mediated microanalysis in a partially filled capillary.

    PubMed

    Zhao, Haiyan; Chen, Zilin

    2013-08-01

    An electrophoretically mediated microanalysis method with partial filling technique was developed for screening aromatase inhibitors in traditional Chinese medicine. The in-capillary enzymatic reaction was performed in 20 mM sodium phosphate buffer (pH 7.4), and sodium phosphate buffer (20 mM, pH 8.0) was used as a background electrolyte. A long plug of coenzyme reduced β-nicotinamide adenine dinucleotide 2'-phosphate hydrate dissolved in the reaction buffer was hydrodynamically injected into a fused silica capillary followed by the injection of reaction buffer, enzyme, and substrate solution. The reaction was initiated with a voltage of 5 kV applied to the capillary for 40 s. The voltage was turned off for 20 min to increase the product amount and again turned on at a constant voltage of 20 kV to separate all the components. Direct detection was performed at 260 nm. The enzyme activity was directly assayed by measuring the peak area of the produced β-nicotinamide adenine dinucleotide phosphate and the decreased peak area indicated the aromatase inhibition. Using the Lineweaver-Burk equation, the Michaelis-Menten constant was calculated to be 50 ± 4.5 nM. The method was applied to the screening of aromatase inhibitors from 15 natural products. Seven compounds were found to have potent AR inhibitory activity.

  12. Long-term treatment of residual or recurrent low-grade endometrial stromal sarcoma with aromatase inhibitors: A report of two cases and a review of the literature

    PubMed Central

    RYU, HYEWON; CHOI, YOON-SEOK; SONG, IK-CHAN; YUN, HWAN-JUNG; JO, DEOG-YEON; KIM, SAMYONG; LEE, HYO JIN

    2015-01-01

    Endometrial stromal sarcoma (ESS) occurs rarely and accounts for only 0.2% of all uterine malignancies. ESS usually expresses estrogen and progesterone receptors, and is regarded as hormone-sensitive. Due to the rarity of these tumors, there are only few case series on the use of aromatase inhibitors in the treatment of low-grade ESS. The present study reports the cases of two patients with residual or recurrent low-grade ESS who experienced long-term disease-free survival following treatment with letrozole. The study also reviews the literature with regard to the data on aromatase inhibitors used in patients with low-grade ESS. In total, 30 patients with recurrent or residual low-grade ESS who were treated with aromatase inhibitors were identified, including the present cases. Among the 30 patients, the overall response rate of advanced low-grade ESS to aromatase inhibitors was 77.4% (complete response, 25.8%; partial response, 51.6%) and the disease control rate was 90.3%. The response rate of first-line treatment was similar to that of second-line therapy or higher (84.6 vs. 72.2%; P=0.453). Duration of aromatase inhibitor treatment ranged from 1.5 to 168 months (median, 26.5 months). The aromatase inhibitors showed minimal adverse effects. In conclusion, aromatase inhibitors, particularly third-generation drugs, are a well-tolerated class of medications that are effective in the treatment of advanced low-grade ESS, with a favorable toxicity profile. PMID:26722331

  13. Germline genetic predictors of aromatase inhibitor concentrations, estrogen suppression and drug efficacy and toxicity in breast cancer patients.

    PubMed

    Hertz, Daniel L; Henry, N Lynn; Rae, James M

    2017-04-01

    The third-generation aromatase inhibitors (AIs), anastrozole, letrozole and exemestane, are highly effective for the treatment of estrogen receptor-positive breast cancer in postmenopausal women. AIs inhibit the aromatase (CYP19A1)-mediated production of estrogens. Most patients taking AIs achieve undetectable blood estrogen concentrations resulting in drug efficacy with tolerable side effects. However, some patients have suboptimal outcomes, which may be due, in part, to inherited germline genetic variants. This review summarizes published germline genetic associations with AI treatment outcomes including systemic AI concentrations, estrogenic response to AIs, AI treatment efficacy and AI treatment toxicities. Significant associations are highlighted with commentary about prioritization for future validation to identify pharmacogenetic predictors of AI treatment outcomes that can be used to inform personalized treatment decisions in patients with estrogen receptor-positive breast cancer.

  14. Enzymic aromatization of 6-alkyl-substituted androgens, potent competitive and mechanism-based inhibitors of aromatase.

    PubMed

    Numazawa, M; Yoshimura, A; Oshibe, M

    1998-01-01

    To gain insight into the relationships between the aromatase inhibitory activity of 6-alkyl-substituted androgens, potent competitive inhibitors, and their ability to serve as a substrate of aromatase, we studied the aromatization of a series of 6alpha- and 6beta-alkyl (methyl, ethyl, n-propyl, n-pentyl and n-heptyl)-substituted androst-4-ene-3,17-diones (ADs) and their androsta-1,4-diene-3,17-dione (ADD) derivatives with human placental aromatase, by gas chromatography-mass spectrometry. Among the inhibitors examined, ADD and its 6alpha-alkyl derivatives with alkyl functions less than three carbons long, together with 6beta-methyl ADD, are suicide substrates of aromatase. All of the steroids, except for 6beta-n-pentyl ADD and its n-heptyl analogue as well as 6beta-n-heptyl AD, were found to be converted into the corresponding 6-alkyl oestrogens. The 6-methyl steroids were aromatized most efficiently in each series, and the aromatization rate essentially decreased in proportion to the length of the 6-alkyl chains in each series, where the 6alpha-alkyl androgens were more efficient substrates than the corresponding 6beta isomers. The Vmax of 6alpha-methyl ADD was approx. 2.5-fold that of the natural substrate AD and approx. 3-fold that of the parent ADD. On the basis of this, along with the facts that the rates of a mechanism-based inactivation of aromatase by ADD and its 6alpha-methyl derivative are similar, it is implied that alignment of 6alpha-methyl ADD in the active site could favour the pathway leading to oestrogen over the inactivation pathway, compared with that of ADD. The relative apparent Km values for the androgens obtained in this study are different from the relative Ki values obtained previously, indicating that there is a difference between the ability to serve as an inhibitor and the ability to serve as a substrate in the 6-alkyl androgen series.

  15. Randomized, Blinded Trial of Vitamin D3 for Treating Aromatase Inhibitor-Associated Musculoskeletal Symptoms (AIMSS)

    PubMed Central

    Shapiro, Alice C.; Adlis, Susan A.; Robien, Kim; Kirstein, Mark N.; Liang, Shuang; Richter, Sara A.; Lerner, Rachel E.

    2017-01-01

    Purpose To evaluate the efficacy and safety of vitamin D3 at 4,000 IU/day as a treatment option for aromatase inhibitor-associated musculoskeletal symptoms (AIMSS) when compared with the usual care dose of 600 IU D3. Methods Single site randomized, double-blind, phase 3 clinical trial in women with AIMSS comparing change in symptoms, reproductive hormones and AI pharmacokinetics. Postmenopausal women ≥18 years with stage I-IIIA breast cancer, taking AI and experiencing AIMSS (Breast Cancer Prevention Trial Symptom Scale-Musculoskeletal Subscale ≥1.5 (BCPT-MS)) were admitted. Following randomization, 116 patients had a run-in period of 1 month on 600 IU D3, then began the randomized assignment to either 600 IU D3 (n=56) or 4,000 IU D3 (n=57) daily for 6 months. The primary endpoint was change in AIMSS from baseline (after 1 month run-in) on the BCPT-MS (general musculoskeletal pain; joint pain; muscle stiffness; range for each question: 0=not at all to 4=extremely). Results Groups had no statistically significant differences demographically or clinically. There were no discernable differences between the randomly allocated treatment groups at 6 months in measures of AIMSS, pharmacokinetics of anastrozole and letrozole, serum levels of reproductive hormones, or adverse events. Conclusions We found no significant changes in AIMSS measures between women who took 4000 IU D3 daily compared with 600 IU D3. The 4000 IU D3 did not adversely affect reproductive hormone levels or the steady state pharmacokinetics of anastrozole or letrozole. In both groups, serum 25(OH)D remained in the recommended range for bone health (≥30 ng/mL) and safety (<50 ng/mL). PMID:26868123

  16. Aromatase Inhibitor-Associated Bone Fractures: A Case-Cohort GWAS and Functional Genomics

    PubMed Central

    Liu, Mohan; Goss, Paul E.; Ingle, James N.; Kubo, Michiaki; Furukawa, Yoichi; Batzler, Anthony; Jenkins, Gregory D.; Carlson, Erin E.; Nakamura, Yusuke; Schaid, Daniel J.; Chapman, Judy-Anne W.; Shepherd, Lois E.; Ellis, Matthew J.; Khosla, Sundeep; Wang, Liewei

    2014-01-01

    Bone fractures are a major consequence of osteoporosis. There is a direct relationship between serum estrogen concentrations and osteoporosis risk. Aromatase inhibitors (AIs) greatly decrease serum estrogen levels in postmenopausal women, and increased incidence of fractures is a side effect of AI therapy. We performed a discovery case-cohort genome-wide association study (GWAS) using samples from 1071 patients, 231 cases and 840 controls, enrolled in the MA.27 breast cancer AI trial to identify genetic factors involved in AI-related fractures, followed by functional genomic validation. Association analyses identified 20 GWAS single nucleotide polymorphism (SNP) signals with P < 5E-06. After removal of signals in gene deserts and those composed entirely of imputed SNPs, we applied a functional validation “decision cascade” that resulted in validation of the CTSZ-SLMO2-ATP5E, TRAM2-TMEM14A, and MAP4K4 genes. These genes all displayed estradiol (E2)-dependent induction in human fetal osteoblasts transfected with estrogen receptor-α, and their knockdown altered the expression of known osteoporosis-related genes. These same genes also displayed SNP-dependent variation in E2 induction that paralleled the SNP-dependent induction of known osteoporosis genes, such as osteoprotegerin. In summary, our case-cohort GWAS identified SNPs in or near CTSZ-SLMO2-ATP5E, TRAM2-TMEM14A, and MAP4K4 that were associated with risk for bone fracture in estrogen receptor-positive breast cancer patients treated with AIs. These genes displayed E2-dependent induction, their knockdown altered the expression of genes related to osteoporosis, and they displayed SNP genotype-dependent variation in E2 induction. These observations may lead to the identification of novel mechanisms associated with fracture risk in postmenopausal women treated with AIs. PMID:25148458

  17. Gonadal development and growth of chickens and turkeys hatched from eggs injected with an aromatase inhibitor.

    PubMed

    Burke, W H; Henry, M H

    1999-07-01

    It was the purpose of these experiments to describe gonadal development and posthatching growth of genetic female chickens and turkeys following in ovo injection of the aromatase inhibitor Fadrazole (CGS 16949A) prior to incubation. In ovo injection of Fadrazole (CGS 16949A) resulted in the development of testes-like gonads in the majority of day-old genetic female chickens and turkey poults. Ninety-eight to 99% of these birds have masculine-type male genitalia at 1 d of age. Microscopic examination of the gonads of day-old genetic female chicks hatched from Fadrazole-treated eggs showed the presence of atypical seminiferous tubules in 3 of 18 individuals and the presence of ovarian follicles in 3 of 18 individuals. No germinal elements were seen in 12 individuals. The gonads in the majority (8/11) of day-old female poults from treated eggs showed the presence of atypical seminiferous tubules. Three of 11 individuals had structures characterized as disorganized or degenerate follicles. Between the day of hatch and 6 wk, gonads in an increasing proportion of female chickens from Fadrazole-treated eggs had normal appearing ovarian follicles. A similar trend was seen in the female turkeys between hatch and 12 wk of age. There were no differences in BW of female chickens hatched from Fadrazole-treated eggs and those from control eggs between the day of hatch and 6 wk of age. The pectoral muscle mass and fat pad weights of these birds did not differ. In one experiment, the BW of female turkeys hatched from Fadrazole-treated eggs was significantly greater than that of controls and equal to that of males at 3 and 6 wk of age. Thereafter, both types of females were of equal weight and significantly lighter than males. Fadrazole treatment did not affect pectoral muscle mass of either sex of turkeys.

  18. Joint pain severity predicts premature discontinuation of aromatase inhibitors in breast cancer survivors

    PubMed Central

    2013-01-01

    Background Premature discontinuation of aromatase inhibitors (AIs) in breast cancer survivors compromises treatment outcomes. We aimed to evaluate whether patient-reported joint pain predicts premature discontinuation of AIs. Methods We conducted a retrospective cohort study of postmenopausal women with breast cancer on AIs who had completed a survey about their symptom experience on AIs with specific measurements of joint pain. The primary outcome was premature discontinuation of AIs, defined as stopping the medication prior to the end of prescribed therapy. Multivariate Cox regression modeling was used to identify predictors of premature discontinuation. Results Among 437 patients who met eligibility criteria, 47 (11%) prematurely discontinued AIs an average of 29 months after initiation of therapy. In multivariate analyses, patient-reported worst joint pain score of 4 or greater on the Brief Pain Inventory (BPI) (Hazard Ratio [HR] 2.09, 95% Confidence Interval [CI] 1.14-3.80, P = 0.016) and prior use of tamoxifen (HR 2.01, 95% CI 1.09-3.70, P = 0.026) were significant predictors of premature discontinuation of AIs. The most common reason for premature discontinuation was joint pain (57%) followed by other therapy-related side effects (30%). While providers documented joint pain in charts for 82% of patients with clinically important pain, no quantitative pain assessments were noted, and only 43% provided any plan for pain evaluation or management. Conclusion Worst joint pain of 4 or greater on the BPI predicts premature discontinuation of AI therapy. Clinicians should monitor pain severity with quantitative assessments and provide timely management to promote optimal adherence to AIs. PMID:24004677

  19. Cell-Based High-Throughput Screening for Aromatase Inhibitors in the Tox21 10K Library.

    PubMed

    Chen, Shiuan; Hsieh, Jui-Hua; Huang, Ruili; Sakamuru, Srilatha; Hsin, Li-Yu; Xia, Menghang; Shockley, Keith R; Auerbach, Scott; Kanaya, Noriko; Lu, Hannah; Svoboda, Daniel; Witt, Kristine L; Merrick, B Alex; Teng, Christina T; Tice, Raymond R

    2015-10-01

    Multiple mechanisms exist for endocrine disruption; one nonreceptor-mediated mechanism is via effects on aromatase, an enzyme critical for maintaining the normal in vivo balance of androgens and estrogens. We adapted the AroER tri-screen 96-well assay to 1536-well format to identify potential aromatase inhibitors (AIs) in the U.S. Tox21 10K compound library. In this assay, screening with compound alone identifies estrogen receptor alpha (ERα) agonists, screening in the presence of testosterone (T) identifies AIs and/or ERα antagonists, and screening in the presence of 17β-estradiol (E2) identifies ERα antagonists. Screening the Tox-21 library in the presence of T resulted in finding 302 potential AIs. These compounds, along with 31 known AI actives and inactives, were rescreened using all 3 assay formats. Of the 333 compounds tested, 113 (34%; 63 actives, 50 marginal actives) were considered to be potential AIs independent of cytotoxicity and ER antagonism activity. Structure-activity analysis suggested the presence of both conventional (eg, 1, 2, 4, - triazole class) and novel AI structures. Due to their novel structures, 14 of the 63 potential AI actives, including both drugs and fungicides, were selected for confirmation in the biochemical tritiated water-release aromatase assay. Ten compounds were active in the assay; the remaining 4 were only active in high-throughput screen assay, but with low efficacy. To further characterize these 10 novel AIs, we investigated their binding characteristics. The AroER tri-screen, in high-throughput format, accurately and efficiently identified chemicals in a large and diverse chemical library that selectively interact with aromatase.

  20. Cell-Based High-Throughput Screening for Aromatase Inhibitors in the Tox21 10K Library

    PubMed Central

    Chen, Shiuan; Hsieh, Jui-Hua; Huang, Ruili; Sakamuru, Srilatha; Hsin, Li-Yu; Xia, Menghang; Shockley, Keith R.; Auerbach, Scott; Kanaya, Noriko; Lu, Hannah; Svoboda, Daniel; Witt, Kristine L.; Merrick, B. Alex; Teng, Christina T.; Tice, Raymond R.

    2015-01-01

    Multiple mechanisms exist for endocrine disruption; one nonreceptor-mediated mechanism is via effects on aromatase, an enzyme critical for maintaining the normal in vivo balance of androgens and estrogens. We adapted the AroER tri-screen 96-well assay to 1536-well format to identify potential aromatase inhibitors (AIs) in the U.S. Tox21 10K compound library. In this assay, screening with compound alone identifies estrogen receptor alpha (ERα) agonists, screening in the presence of testosterone (T) identifies AIs and/or ERα antagonists, and screening in the presence of 17β-estradiol (E2) identifies ERα antagonists. Screening the Tox-21 library in the presence of T resulted in finding 302 potential AIs. These compounds, along with 31 known AI actives and inactives, were rescreened using all 3 assay formats. Of the 333 compounds tested, 113 (34%; 63 actives, 50 marginal actives) were considered to be potential AIs independent of cytotoxicity and ER antagonism activity. Structure-activity analysis suggested the presence of both conventional (eg, 1, 2, 4, - triazole class) and novel AI structures. Due to their novel structures, 14 of the 63 potential AI actives, including both drugs and fungicides, were selected for confirmation in the biochemical tritiated water-release aromatase assay. Ten compounds were active in the assay; the remaining 4 were only active in high-throughput screen assay, but with low efficacy. To further characterize these 10 novel AIs, we investigated their binding characteristics. The AroER tri-screen, in high-throughput format, accurately and efficiently identified chemicals in a large and diverse chemical library that selectively interact with aromatase. PMID:26141389

  1. Reduced estradiol synthesis by letrozole, an aromatase inhibitor, is protective against development of pentylenetetrazole-induced kindling in mice.

    PubMed

    Rashid, Davood; Panda, B P; Vohora, Divya

    2015-11-01

    Neurosteroids, such as testosterone and their metabolites, are known to modulate neuronal excitability. The enzymes regulating the metabolism of these neurosteroids, thus, may be targeted as a noval strategy for the development of new antiepileptic drugs. The present work targeted two such enzymes i,e aromatase and 5α-reductase in order to explore the potential of letrozole (an aromatase inhibitor) on pentylenetetrazole (PTZ)-induced kindling in mice and the ability of finasteride (a 5α-reductase inhibitor) to modulate any such effects. PTZ (30 mg/kg, i.p.), when administered once every two days (for a total of 24 doses) induced kindling in Swiss albino mice. Letrozole (1 mg/kg, p.o.), administered prior to PTZ, significantly reduced the % incidence of kindling, delayed mean onset time of seizures and reduced seizure severity score. Letrozole reduced the levels of plasma 17β-estradiol after induction of kindling. The concurrent administration of finasteride and letrozole produced effects similar to letrozole on PTZ-kindling and on estradiol levels. This implies that the ability of letrozole to redirect the synthesis of dihydrotestosterone (DHT) and 5α-androstanediol from testosterone doesn't appear to play a significant role in the protective effects of letrozole against PTZ kindling. Letrozole, however, increased the levels of 5α-DHT in mice plasma. The aromatase inhibitors, thus, may be exploited for inhibiting the synthesis of proconvulsant (17β-estradiol) and/or redirecting the synthesis of anticonvulsant (DHT and 5α-androstanediol) neurosteroids.

  2. Prognostic impact of progesterone receptor status combined with body mass index in breast cancer patients treated with adjuvant aromatase inhibitor.

    PubMed

    Ohara, Masahiro; Akimoto, Etsushi; Noma, Midori; Matsuura, Kazuo; Doi, Mihoko; Kagawa, Naoki; Itamoto, Toshiyuki

    2015-11-01

    Aromatase inhibitors have played a central role in endocrine therapy for the treatment of estrogen receptor (ER)-positive breast cancer in postmenopausal patients. However, prognostic factors for recurrence following such treatment have not been identified. The current study aimed to validate the prognostic value of endocrine-related progesterone receptor (PgR) status combined with body mass index (BMI). Among 659 consecutive patients with primary breast cancer who underwent curative surgery between 2002 and 2012, 184 postmenopausal patients with ER-positive (ER+) and human epidermal growth factor receptor type 2-negative (HER2-) breast cancer who were treated with adjuvant aromatase inhibitor therapy were assessed. The patients were assigned to groups based on BMI, according to the WHO cut-off value: ≥25 kg/m(2) (high, H) or <25 kg/m(2) (low, L). Positive nodal status, negative PgR status, BMI-H and a high Ki-67 labeling index (≥20%) were found to be significantly associated with a short recurrence-free interval (RFI) upon univariate analysis (P=0.048, 0.007, 0.027, and 0.012, respectively). The patients were further grouped based on their combined PgR/BMI status. The RFI was significantly shorter in the PgR- and/or BMI-H group compared with that of the PgR+/BMI-L group (P=0.012). Multivariate analysis revealed PgR- tumors and/or BMI-H and positive nodal status to be independent prognostic factors (P=0.012 and 0.020, respectively). The present findings indicate that PgR/BMI status may serve as a practical tool in the management of ER+ and HER2- breast cancer in patients treated with adjuvant aromatase inhibitors.

  3. Synthesis and evaluation of benzoxazolinonic imidazoles and derivatives as non-steroidal aromatase inhibitors.

    PubMed

    Nativelle-Serpentini, Celine; Moslemi, Safa; Yous, Said; Park, Chang Ha; Lesieur, Daniel; Sourdaine, Pascal; Séralini, Gilles-Eric

    2004-04-01

    New compounds were tested in vitro on aromatase activity in human placental and equine testicular microsomes. Equine aromatase, very well characterized biochemically, is used as a comparative model to understand the mechanism of aromatase inhibition. Among 15 molecules screened, 5 of them (11-15) strongly inhibit human and equine aromatases with IC50 values ranging from 13-85nM and from 23-103nM respectively. These results were corroborated by Ki/Km values. Moreover, spectral studies showed a type II spectrum with both enzymes, which is characteristic of an interaction between the nitrogen atom of the molecule and the heme of the cytochrome P450. Compound 12, which has the lowest IC50 and Ki/Km ratio, inactivates aromatase in a dose and time-dependent manner. This might be very important for the treatment of estrogen-dependent diseases such as breast cancer. Finally, MTT assays on E293 cells revealed that the molecules were not cytotoxic.

  4. Direct Effects, Compensation, and Recovery in Female Fathead Minnows Exposed to the Aromatase Inhibitor Fadrozole

    EPA Science Inventory

    A variety of chemicals present in the environment have the potential to inhibit aromatase, an enzyme critical to estrogen synthesis. The objective of this study was to provide a detailed characterization of the molecular and biochemical responses of female fathead minnows to a m...

  5. Sex Amphibian, Xenopus tropicalis, following Larval Exposure to an Aromatase Inhibitor

    EPA Science Inventory

    Aromatase is a steroidogenic enzyme that catalyzes the conversion of androgens to estrogens in vertebrates. Modulation of this enzyme’s activity by xenobiotic exposure has been shown to adversely affect gonadal differentiation in a number of diverse species. We hypothesized tha...

  6. Treatment of idiopathic short stature: effects of gonadotropin-releasing hormone analogs, aromatase inhibitors and anabolic steroids.

    PubMed

    Dunkel, Leo

    2011-01-01

    Modulation of sex steroid action on the growth plate can, at least theoretically, increase adult height in children and adolescents with idiopathic short stature. Gonadotropin-releasing hormone (GnRH) analog therapy during adolescence has been shown effective in a placebo-controlled study, but to obtain clinically significant increases in adult height, the treatment duration must be lengthy (several years). Furthermore, such treatment seems to compromise bone health and, because of the resulting delay in pubertal development, likely has psychosocial consequences. Therefore, GnRH analogs are no longer recommended to augment height in adolescents with short stature and normally timed puberty. Aromatase inhibitors are probably more effective than GnRH analogs in promoting increased adult height in children with short stature and, unlike GnRH analogs, do not delay pubertal development in males. However, due to a dearth of safety data with aromatase inhibitors for the treatment of short stature, their use outside a research setting is currently not recommended. Positive effects of anabolic steroids on adult height have not been documented.

  7. Synthesis and biochemical studies of 7 alpha-substituted androsta-1,4-diene-3,17-diones as enzyme-activated irreversible inhibitors of aromatase.

    PubMed

    Ebrahimian, S; Chen, H H; Brueggemeier, R W

    1993-09-01

    Several 7 alpha-thiosubstituted derivatives of androstenedione have demonstrated effective inhibition of aromatase, the cytochrome P450 enzyme complex responsible for the biosynthesis of estrogens. Introduction of an additional double bond in the A ring resulted in 7 alpha-(4'-amino)phenylthioandrosta-1,4-diene-3,17-dione (7 alpha-APTADD), a potent inhibitor that inactivated aromatase by an enzyme-catalyzed process. Additional 7 alpha-thiosubstituted androsta-1,4-diene-3,17-dione derivatives were designed to further examine enzyme-catalyzed inactivation. Two halogenated and one unsubstituted 7 alpha-phenylthioandrosta-1,4-diene-3,17-diones were synthesized via an acid-catalyzed conjugate Michael addition of substituted thiophenols with androsta-1,4,6-triene-3,17-dione. Two 7 alpha-naphthylthioandrosta-1,4-diene-3,17-diones were synthesized via either acid-catalyzed or based-catalyzed conjugate Michael addition of substituted thionaphthols with androsta-1,4,6-triene-3,17-dione. These agents were evaluated for aromatase inhibitory activity in the human placental microsomal preparation. Under initial velocity assay conditions of low product formation, the inhibitors demonstrated potent inhibition of aromatase, with apparent Ki's ranging from 12 to 27 nM. Furthermore, these compounds produced time-dependent, first-order inactivation of aromatase in the presence of NADPH, whereas no aromatase inactivation was observed in the absence of NADPH. This enzyme-activated irreversible inhibition, also referred to as mechanism-based inhibition, can be prevented by the substrate androstenedione. Thus, the apparent Ki values for these inhibitors are consistent with earlier studies on 7 alpha-substituted competitive inhibitors that indicate bulky substituents can be accommodated at the 7 alpha-position.(ABSTRACT TRUNCATED AT 250 WORDS)

  8. Aromatase inhibitors block natural sex change and induce male function in the protandrous black porgy, Acanthopagrus schlegeli Bleeker: possible mechanism of natural sex change.

    PubMed

    Lee, Yan-Horn; Yueh, Wen-Shiun; Du, Jin-Lien; Sun, Lian-Tien; Chang, Ching-Fong

    2002-06-01

    The objectives of the present study were to investigate the effects of oral administration of aromatase inhibitors on sex change, milt volume, 11-ketotestosterone (11-KT), and LH in plasma; aromatase activity in gonad, pituitary, and brain in the protandrous fish, black porgy (Acanthopagus schlegeli Bleeker). Two-year-old functional male black porgy were divided into two groups; one was fed a control diet and the other was fed a diet mixed with aromatase inhibitors (AIs; fadrozole and 1,4,6-androstatriene-3,17-dione, each 10 mg/kg feed) for 8.5 mo. A significantly higher gonadosomatic index was observed in the AI group. Fish treated with AIs showed complete suppression of natural sex change. Significantly higher levels of plasma 11-KT, LH, and milt volume were shown in the AI group than the controls. Lower aromatase activity in the gonad, pituitary, forebrain, midbrain, and hindbrain in concordance with the suppression of sex change was observed in the AI group. The data show that aromatase is directly involved in the mechanism of natural sex change of protandrous black porgy. AIs also enhanced male function in concordance with the elevated plasma levels of 11-KT and spermiation in milt volume.

  9. Testosterone vs. aromatase inhibitor in older men with low testosterone: effects on cardiometabolic parameters.

    PubMed

    Dias, J P; Shardell, M D; Carlson, O D; Melvin, D; Caturegli, G; Ferrucci, L; Chia, C W; Egan, J M; Basaria, S

    2017-01-01

    Testosterone (T) replacement is being increasingly offered to older men with age-related decline in testosterone levels. The effects of long-term testosterone replacement and aromatase inhibition (AI) on glucose homeostasis and cardiometabolic markers were determine in older non-diabetic men with low testosterone levels. Men ≥65 years, mean age 71 ± 3 years with serum total T < 350 ng/dL were randomized in a double-blind, placebo-controlled, parallel-group, proof-of-concept trial evaluating the effects of 5 g transdermal testosterone gel (TT) (n = 10), 1 mg anastrozole (n = 10) or placebo (n = 9) daily for 12 months. Homeostatic Model Assessment of insulin resistance (HOMAIR ) was the primary outcome. Secondary outcomes included OGIS in response to OGTT, fasting lipids, C-reactive protein (CRP), adipokines, and abdominal and mid-thigh fat by computed tomography. All outcomes were assessed at baseline and 12 months. After 12 months, absolute changes in HOMAIR in both treatment arms (TT group: -0.05 ± 0.21); (AI group: 0.15 ± 0.10) were similar to placebo (-0.11 ± 0.26), as were CRP and fasting lipid levels. Adiponectin levels significantly decreased in the TT group (-1.8 ± 0.9 mg/L, p = 0.02) and abdominal subcutaneous fat (-60.34 ± 3.19 cm(2) , p = 0.003) and leptin levels (-1.5 ± 1.2 ng/mL, p = 0.04) were significantly lower with AI. Mid-thigh subcutaneous fat was reduced in both treatment arms (TT group: -4.88 ± 1.24 cm(2) , p = 0.008); (AI group: -6.05 ± 0.87 cm(2) , p = 0.0002). In summary, in this proof-of-concept trial, changes in HOMAIR AI were similar in all three groups while the effects of intervention on subcutaneous fat distribution and adipokines were variable. Larger efficacy and safety trials are needed before AI pharmacotherapy can be considered as a treatment option for low T levels in older men.

  10. Aromatase and breast cancer.

    PubMed

    Brodie, A; Sabnis, G; Jelovac, D

    2006-12-01

    Several aromatase inhibitors and also new antiestrogens are now available for treating breast cancer. We have developed a model to compare the antitumor efficacy of these agents and to explore strategies for their optimal use. Results from the model have been predictive of clinical outcome. In this model, tumors are grown in ovariectomized, immunodeficient mice from MCF-7 human breast cancer cells transfected with the aromatase gene (MCF-7Ca). The possibility that blockade of estrogen action and estrogen synthesis may be synergistic was explored by treating mice with the aromatase inhibitor letrozole and the antiestrogen tamoxifen alone and in combination. The results indicated that letrozole alone was better than all other treatments. In addition, when tamoxifen treatment was no longer effective, tumor growth was significantly reduced in mice switched to letrozole treatment. However, tumors ultimately began to grow during continued treatment. To investigate the mechanisms by which tumors eventually adapt and grow during letrozole treatment, we determined the expression of signaling proteins in tumors during the course of letrozole treatment compared to the tumors of control mice. Tumors initially up-regulated the ER while responding to treatment, but subsequently receptor levels decreased in tumors unresponsive to letrozole. Also, Her-2 and adapter proteins (p-Shc and Grb-2) as well as all of the signaling proteins in the MAPK cascade (p-Raf, p-Mekl/2, and p-MAPK), but not in the Pl3/Akt pathway, were increased in tumors no longer responsive to letrozole. To investigate whether sensitivity to letrozole could be regained, cells were isolated from the letrozole resistant tumors (LTLT) and treated with inhibitors of the MAPKinase pathway (PD98059 and UO126). These compounds reduced MAPK activity and increased ER expression. EGFR/Her-2 inhibitors, gefitinib and AEE78S although not effective in the parental MCF-70a cells, restored the sensitivity of LTLT cells to

  11. Altered Gene Expression in the Brain and Ovaries of Zebrafish Exposed to the Aromatase Inhibitor Fadrosole: Microarray Analysis for Hypothesis Generation

    EPA Science Inventory

    A part of an overall program of research aimed at examining system-wide responses of the hypothalamic-pituitary-gonadal axis in fish to endocrine active chemicals acting through a variety of modes of action, we exposed zebrafish (Danio rerio) to the aromatase inhibitor fadrozole ...

  12. Altered Gene Expression in the Brain and Ovaries of Zebrafish Exposed to the Aromatase Inhibitor Fadrozole: Microarray analysis and Hypothesis Generation

    EPA Science Inventory

    As part of a research effort examining system-wide responses of the hypothalamic-pituitary-gonadal (HPG) axis in fish to endocrine active chemicals (EACs) with different modes of action, we exposed zebrafish (Danio rerio) to 25 or 100 ìg/L of the aromatase inhibitor fadrozole for...

  13. Binding mode of triazole derivatives as aromatase inhibitors based on docking, protein ligand interaction fingerprinting, and molecular dynamics simulation studies

    PubMed Central

    Mojaddami, Ayyub; Sakhteman, Amirhossein; Fereidoonnezhad, Masood; Faghih, Zeinab; Najdian, Atena; Khabnadideh, Soghra; Sadeghpour, Hossein; Rezaei, Zahra

    2017-01-01

    Aromatase inhibitors (AIs) as effective candidates have been used in the treatment of hormone-dependent breast cancer. In this study, we have proposed 300 structures as potential AIs and filtered them by Lipinski's rule of five using DrugLito software. Subsequently, they were subjected to docking simulation studies to select the top 20 compounds based on their Gibbs free energy changes and also to perform more studies on the protein-ligand interaction fingerprint by AuposSOM software. In this stage, anastrozole and letrozole were used as positive control to compare their interaction fingerprint patterns with our proposed structures. Finally, based on the binding energy values, one active structure (ligand 15) was selected for molecular dynamic simulation in order to get information for the binding mode of these ligands within the enzyme cavity. The triazole of ligand 15 pointed to HEM group in aromatase active site and coordinated to Fe of HEM through its N4 atom. In addition, two π-cation interactions was also observed, one interaction between triazole and porphyrin of HEM group, and the other was 4-chloro phenyl moiety of this ligand with Arg115 residue. PMID:28255310

  14. From nonsteroidal aromatase inhibitors to multifunctional drug candidates: classic and innovative strategies for the treatment of breast cancer.

    PubMed

    Gobbi, Silvia; Cavalli, Andrea; Bisi, Alessandra; Recanatini, Maurizio

    2008-01-01

    Aromatase is the enzyme responsible for the conversion of androgens to estrogens and represents the main source of local estrogens in post-menopausal breast cancer tissue. Nonsteroidal aromatase inhibitors (NSAIs) are able to reduce growth-stimulatory effects of estrogens in hormone-dependent breast cancer, and third generation NSAIs are currently approved as first-line therapy for the treatment of postmenopausal women with metastatic advanced breast cancer. Nevertheless, some issues in this area still need to be addressed and research efforts are aimed both at identifying new molecules of therapeutic interest and at exploring different options for the modulation of this enzyme. In this review, an update of the latest developments in the field of NSAIs is presented, to provide a broad view on the recent progress in this area. Beside classical structure-activity relationships studies and development of natural product derivatives, rational approaches for both ligand- and structure-based design are described. Moreover, novel strategies for the development of multitarget-directed molecules are also presented. Finally, some possible future developments in this research area are briefly considered.

  15. [Criteria for evaluating the effectiveness of aromatase inhibitors in the neoadjuvant treatment of patients with endometrial carcinoma].

    PubMed

    Bershteĭn, L M; Danilova, M A; Kovalevskiĭ, A Iu; Gershfel'd, E D; Poroshina, T E; Tsyrlina, T E; Meshkova, I E; Turkevich, E A; Maksimov, S Ia

    2009-01-01

    Clinical and experimental effects of neoadjuvant treatment of endometrial cancer patients with non-steroidal aromatase inhibitors: letrozole (femara, n=10, 2.5 mg/day, 14 days), anastrozole (arimidex, n=15,1 mg/day, 28 days) and exemestane (aromazine, n=13, 25 mg/day, 14 days) were compared. Administration of anastrozole was mostly frequently followed by pain relief in the lower abdomen and/or decreased rates of uterine discharge. Endometrial wall thickness (M-echo signal) decreased significantly in 60% of patients receiving anastrozole, exemestane - 58.3% and letrozole - 40%. Substantial drop in intratumoral aromatase and blood estradiol levels occurred more frequently after anastrozole and letrozole while progesterone receptor levels in tumor were markedly lower after exemestane administration. Assay of blood LH (except letrozole), FSH and cholesterol appeared to be of less relevance. On the contrary, significance of assessment of marker Ki-67 expression, which, in the case of anastrozole, dropped in 6 out of 12 patients after a 28-day course, could hardly be underestimated.

  16. The effect of the aromatase inhibitor, 4-(phenylthio)-4-androstene-3,17-dione, on dimethylbenz(A)anthracene-induced rat mammary tumors.

    PubMed

    Abul-Hajj, Y J

    1989-01-01

    4-(Phenylthio)-4-androstene-3,17-dione (4-PTAD), a known inhibitor of human placental aromatase, was examined as a growth inhibitor of DMBA-induced rat mammary tumors. Subcutaneous administration of 4-PTAD at dose levels of 25 or 50 mg/kg/day caused a significant decrease in hormone-dependent tumor growth. Resumption of tumor growth occurred when either the administration of inhibitor was stopped or when inhibitor was coadministered with estradiol indicating that suppression of tumor growth was due to inhibition of estrogen biosynthesis. Additionally, plasma levels of estradiol were found to be lower in the animals treated with 4-PTAD. The major metabolite of 4-PTAD in vitro was identified as 4-(phenylthio)-4-androstene-17 beta-ol-3-one and was found to have 60% of the aromatase inhibitory activity of 4-PTAD.

  17. Steroidal and non-steroidal third-generation aromatase inhibitors induce pain-like symptoms via TRPA1.

    PubMed

    Fusi, Camilla; Materazzi, Serena; Benemei, Silvia; Coppi, Elisabetta; Trevisan, Gabriela; Marone, Ilaria M; Minocci, Daiana; De Logu, Francesco; Tuccinardi, Tiziano; Di Tommaso, Maria Rosaria; Susini, Tommaso; Moneti, Gloriano; Pieraccini, Giuseppe; Geppetti, Pierangelo; Nassini, Romina

    2014-12-08

    Use of aromatase inhibitors (AIs), exemestane, letrozole and anastrozole, for breast cancer therapy is associated with severe pain symptoms, the underlying mechanism of which is unknown. The electrophilic nature of AIs suggests that they may target the transient receptor potential ankyrin 1 (TRPA1) channel, a major pathway in pain transmission and neurogenic inflammation. AIs evoke TRPA1-mediated calcium response and current in rodent nociceptors and human cells expressing the recombinant channel. In mice, AIs produce acute nociception, which is exaggerated by pre-exposure to proalgesic stimuli, and, by releasing sensory neuropeptides, neurogenic inflammation in peripheral tissues. AIs also evoke mechanical allodynia and decreased grip strength, which do not undergo desensitization on prolonged AI administration. These effects are markedly attenuated by TRPA1 pharmacological blockade or in TRPA1-deficient mice. TRPA1 is a major mediator of the proinflammatory/proalgesic actions of AIs, thus suggesting TRPA1 antagonists for the treatment of pain symptoms associated with AI use.

  18. Association of functional polymorphisms in CYP19A1 with aromatase inhibitor associated arthralgia in breast cancer survivors

    PubMed Central

    2011-01-01

    Introduction Aromatase inhibitor-associated arthralgia (AIAA) is a common and often debilitating symptom in breast cancer survivors. Since joint symptoms have been related to estrogen deprivation through the menopausal transition, we hypothesized that genetic polymorphisms in CYP19A1, the final enzyme in estrogen synthesis, may be associated with the occurrence of AIAA. Methods We performed a cross-sectional study of postmenopausal women with stage 0 to III breast cancer receiving adjuvant aromatase inhibitor (AI) therapy. Patient-reported AIAA was the primary outcome. DNA was genotyped for candidate CYP19A1 polymorphisms. Serum estrogen levels were evaluated by radioimmunoassay. Multivariate analyses were performed to examine associations between AIAA and genetic variants controlling for possible confounders. Results Among 390 Caucasian participants, 50.8% reported AIAA. Women carrying at least one 8-repeat allele had lower odds of AIAA (adjusted odds ratio (AOR) 0.41, 95% confidence interval (CI) 0.21 to 0.79, P = 0.008) after adjusting for demographic and clinical covariates. Estradiol and estrone were detectable in 47% and 86% of subjects on AIs, respectively. Although these post-AI levels were associated with multiple genotypes, they were not associated with AIAA. In multivariate analyses, women with more recent transition into menopause (less than five years) were significantly more likely to report AIAA than those greater than ten years post-menopause (AOR 3.31, 95% CI 1.72 to 6.39, P < 0.001). Conclusions Functional polymorphism in CYP19A1 and time since menopause are associated with patient-reported AIAA, supporting the hypothesis that the host hormonal environment contributes to the pathophysiology of AAIA. Prospective investigation is needed to further delineate relationships between host genetics, changing estrogen levels and AIAA. PMID:21251330

  19. New aromatase inhibitors. Synthesis and biological activity of pyridyl-substituted tetralone derivatives.

    PubMed

    Bayer, H; Batzl, C; Hartmann, R W; Mannschreck, A

    1991-09-01

    The (E)-2-(4-pyridylmethylene)-1-tetralones 1-7 (1, H; 2, 5-OCH3; 3, 6-OCH3; 4, 7-OCH3; 5, 5-OH; 6, 6-OH; 7, 7-OH) were obtained by aldol condensation of the corresponding 1-tetralones with 4-pyridinecarboxaldehyde, and in the case of the OH compounds 5 and 7 subsequent ether cleavage of the OCH3-substituted 2-(4-pyridylmethylene)-1-tetralones. Catalytic hydrogenation of 1-4 gave the 2-(4-pyridylmethyl)-1-tetralones 8-11 (8, H; 9, 5-OCH3; 10, 6-OCH3; 11, 7-OCH3). Subsequent ether cleavage of 9-11 led to the corresponding OH compounds 12-14 (12, 5-OH; 13, 6-OH; 14, 7-OH). The enantiomers of 11 and 12 were separated semipreparatively by HPLC on triacetylcellulose. All compounds (1-14) showed an inhibition of human placental aromatase exhibiting relative potencies from 2.2 to 213 [compounds 6 and (+)-12, respectively; aromatase inhibitory potency of aminoglutethimide (AG) = 1]. The compounds exhibited no or only a weak inhibition of desmolase [cholesterol side chain cleavage enzyme; maximum activity shown by 12, 23% inhibition (25 microM); AG, 53% inhibition (25 microM)]. In vivo, however, the compounds were not superior to AG as far as the reduction of the plasma estradiol concentration and the mammary carcinoma (MC) inhibiting properties are concerned (PMSG-primed SD rats as well as DMBA-induced MC of the SD rat, pre- and postmenopausal experiments, and the transplantable MXT-MC of the BD2F1 mouse). This is due to a fast decrease of the plasma E2 concentration inhibiting effect as could be shown by a kinetic experiment. In addition, select compounds inhibited rat ovarian aromatase much less than human placental aromatase (12, factor of 10). Estrogenic effects as a cause for the poor in vivo activity of the test compounds could be excluded, since they did not show affinity for the estrogen receptor.

  20. MCF-7aro/ERE, a novel cell line for rapid screening of aromatase inhibitors, ERalpha ligands and ERRalpha ligands.

    PubMed

    Lui, Ki; Tamura, Takaya; Mori, Taisuke; Zhou, Dujin; Chen, Shiuan

    2008-07-15

    We have previously generated a breast cancer cell line, MCF-7aro, which over-expresses aromatase and is also ER positive. Recently, this MCF-7aro cell line was stably transfected with a promoter reporter plasmid, pGL3-Luc, containing three repeats of estrogen responsive element (ERE). Experiments using MCF-7aro/ERE have demonstrated that it is a novel, non-radioactive screening system for aromatase inhibitors (AIs), ERalpha ligands and ERRalpha ligands. The screening is carried out in a 96-well plate format. To evaluate this system, the cells were cultured overnight in charcoal-dextran stripped FBS medium supplemented with 0.1 nM testosterone or 17beta-estradiol, and various concentrations of antiestrogens or AIs. We found that the luciferase activity was induced when the cells were cultured either in the presence of testosterone or 17beta-estradiol. Furthermore, a 50% decrease in luciferase activity could be achieved when the cells were cultured in the presence of testosterone together with letrozole, anastrozole, tamoxifen or fulvestrant (concentrations being 75 nM, 290 nM, 100 nM, and 5 nM, respectively), compared to the testosterone-only cultured cells. Using this assay system, we confirmed that 3(2'-chlorophenyl)-7-methoxy-4-phenylcoumarin is an agonist of ER. Furthermore, genestein has been shown to be a ligand of ERRalpha because its binding could be blocked by an ERRalpha inverse agonist, XCT790. These results indicate that MCF-7aro/ERE is a novel cell line for rapid screening of AIs, ERalpha ligands and ERRalpha ligands.

  1. Use of Aromatase Inhibitors in Large Cell Calcifying Sertoli Cell Tumors: Effects on Gynecomastia, Growth Velocity, and Bone Age

    PubMed Central

    Crocker, Melissa K.; Gourgari, Evgenia; Stratakis, Constantine A.

    2014-01-01

    Context: Large cell calcifying Sertoli cell tumors (LCCSCT) present in isolation or, especially in children, in association with Carney Complex (CNC) or Peutz-Jeghers Syndrome (PJS). These tumors overexpress aromatase (CYP19A1), which leads to increased conversion of delta-4-androstenedione to estrone and testosterone to estradiol. Prepubertal boys may present with growth acceleration, advanced bone age, and gynecomastia. Objective: To investigate the outcomes of aromatase inhibitor therapy (AIT) in prepubertal boys with LCCSCTs. Design: Case series of a very rare tumor and chart review of cases treated at other institutions. Setting: Tertiary care and referral center. Patients: Six boys, five with PJS and one with CNC, were referred to the National Institutes of Health for treatment of LCCSCT. All patients had gynecomastia, testicular enlargement, and advanced bone ages, and were being treated by their referring physicians with AIT. Interventions: Patients were treated for a total of 6–60 months on AIT. Main Outcome Measures: Height, breast tissue mass, and testicular size were all followed; physical examination, scrotal ultrasounds, and bone ages were obtained, and hormonal concentrations and tumor markers were measured. Results: Tumor markers were negative. All patients had decreases in breast tissue while on therapy. Height percentiles declined, and predicted adult height moved closer to midparental height as bone age advancement slowed. Testicular enlargement stabilized until entry into central puberty. Only one patient required unilateral orchiectomy. Conclusions: Patients with LCCSCT benefit from AIT with reduction and/or elimination of gynecomastia and slowing of linear growth and bone age advancement. Further study of long-term outcomes and safety monitoring are needed but these preliminary data suggest that mammoplasty and/or orchiectomy may be foregone in light of the availability of medical therapy. PMID:25226294

  2. Data of aromatase inhibitors alone and in combination with raloxifene on microarchitecture of lumbar vertebrae and strength test in femoral diaphysis of VCD treated ovotoxic mice.

    PubMed

    Kalam, Abul; Talegaonkar, Sushama; Vohora, Divya

    2017-02-01

    Currently, the third generation aromatase inhibitors are the drugs of choice for treatment of early and advanced breast cancer in postmenopausal women. The negative impact of these drugs on bone health is the significant limiting factor during this therapy. Here we report the effect of two aromatase inhibitors viz. letrozole and exemestane alone and in combination with raloxifene on lumbar vertebrae and femoral diaphysis after one month of treatment but no discernible effects were observed on bone when tested by micro CT and strength test except in trabecular number which was reduced in lumbar vertebrae following letrozole and exemestane. Further studies with letrozole and exemestane should be done at higher doses for longer duration of time to check whether effects are observed in other parameters as well. The data is an extension of our published work in Mol. Cell Endocrinology (A. Kalam, S. Talegaonkar, D. Vohora, 2017) [1] describing letrozole-induced bone loss on femoral epiphysis and its reversal by raloxifene.

  3. Aromatase inhibition by bioavailable methylated flavones.

    PubMed

    Ta, Nga; Walle, Thomas

    2007-10-01

    Previous studies have shown chrysin, 7-hydroxyflavone and 7,4'-dihydroxyflavone to be the most potent flavonoid inhibitors of aromatase. However, very poor oral bioavailability is a major limitation for the successful use of dietary flavonoids as chemopreventive agents. We have recently shown that methylated flavones, including 5,7-dimethoxyflavone, 7-methoxyflavone and 7,4'-dimethoxyflavone, are much more resistant to metabolism than their unmethylated analogs and have much higher intestinal absorption. In this study, we examined these fully methylated flavones as potential aromatase inhibitors for the prevention and/or treatment of hormone-dependent cancers. Whereas 5,7-dimethoxyflavone had poor effect compared to its unmethylated analog chrysin, 7-methoxyflavone and 7,4'-dimethoxyflavone were almost equipotent to their unmethylated analogs with IC(50) values of 2-9 microM. Thus, some fully methylated flavones appear to have great potential as cancer chemopreventive/chemotherapeutic agents.

  4. Aromatase inhibition by bioavailable methylated flavones

    PubMed Central

    Ta, Nga; Walle, Thomas

    2007-01-01

    Previous studies have shown chrysin, 7-hydroxyflavone and 7,4′-dihydroxyflavone to be the most potent flavonoid inhibitors of aromatase. However, very poor oral bioavailability is a major limitation for the successful use of dietary flavonoids as chemopreventive agents. We have recently shown that methylated flavones, including 5,7-dimethoxyflavone, 7-methoxyflavone and 7,4′-dimethoxyflavone, are much more resistant to metabolism than their unmethylated analogs and have much higher intestinal absorption. In this study, we examined these fully methylated flavones as potential aromatase inhibitors for the prevention and/or treatment of hormone-dependent cancers. Whereas 5,7-dimethoxyflavone had poor effect compared to its unmethylated analog chrysin, 7-methoxyflavone and 7,4′-dimethoxyflavone were almost equipotent to their unmethylated analogs with IC50 values of 2 to 9 μM. Thus, some fully methylated flavones appear to have great potential as cancer chemopreventive/chemotherapeutic agents. PMID:17624765

  5. Ethinylestradiol is beneficial for postmenopausal patients with heavily pre-treated metastatic breast cancer after prior aromatase inhibitor treatment: a prospective study

    PubMed Central

    Iwase, H; Yamamoto, Y; Yamamoto-Ibusuki, M; Murakami, K-I; Okumura, Y; Tomita, S; Inao, T; Honda, Y; Omoto, Y; Iyama, K-I

    2013-01-01

    Background: Oestrogens usually stimulate the progression of oestrogen receptor (ER)-positive breast cancer. Paradoxically, high-dose oestrogens suppress the growth of these tumours in certain circumstances. Methods: We prospectively examined the efficacy and safety of ethinylestradiol treatment (3 mg per day oral) in postmenopausal patients with advanced or recurrent ER-positive breast cancer who had previously received endocrine therapies, especially those with resistance to aromatase inhibitors. Results: Eighteen patients were enrolled with the median age of 63 years and the mean observation time of 9.2 months. Three cases withdrew within 1 week due to oestrogen flare reactions with nausea, fatigue and muscle-skeletal pain. The response rate was 50% (9 out of 18), and the clinical benefit rate was 56% (10 out of 18). The stable disease (<6 months) was 17% (3 out of 18) and another 2 cases were judged as progressive disease. Time-to-treatment failure including 2 on treatment was a median of 5.6 months (range 0.1 to 14.5+). Although vaginal bleeding or endometrial thickening was observed in patients receiving long-term treatment, there were no severe adverse events, such as deep venous thrombosis or other malignancies. Conclusion: Although the mechanism of this treatment has not been fully understood, our data may contribute to change the common view of late-stage endocrine therapy. PMID:24002591

  6. A molecular model for the interaction between vorozole and other non-steroidal inhibitors and human cytochrome P450 19 (P450 aromatase).

    PubMed

    Koymans, L M; Moereels, H; Vanden Bossche, H

    1995-06-01

    In a previous study (Vanden Bossche et al., Breast Cancer Res. Treat. 30 (1994) 43) the interaction between (+)-S-vorozole and the I-helix of cytochrome P450 19 (P450 aromatase) has been reported. In the present study we extended the "I-helix model" by incorporating the C-terminus of P450 aromatase. The crystal structures of P450 101 (P450 cam), 102 (P450 BM-3) and 108 (P450 terp) reveal that the C-terminus is structurally conserved and forms part of their respective substrate binding pocket. Furthermore, the present study is extended to the interaction between P450 aromatase and its natural substrate androstenedione and the non-steroidal inhibitors (-)-R-vorozole, (-)-S-fadrozole, R-liarozole and (-)-R-aminoglutethimide. It is found that (+)-S-vorozole, (-)-S-fadrozole and R-liarozole bind in a comparable way to P450 aromatase and interact with both the I-helix (Glu302 and Asp309) and C-terminus (Ser478 and His480). The weak activity of (-)-R-aminoglutethimide might be attributed to a lack of interaction with the C-terminus.

  7. Effects of selective serotonin reuptake inhibitors on three sex steroids in two versions of the aromatase enzyme inhibition assay and in the H295R cell assay.

    PubMed

    Jacobsen, Naja Wessel; Hansen, Cecilie Hurup; Nellemann, Christine; Styrishave, Bjarne; Halling-Sørensen, Bent

    2015-10-01

    Selective serotonin reuptake inhibitors are known to have a range of disorders that are often linked to the endocrine system e.g. hormonal imbalances, breast enlargement, sexual dysfunction, and menstrual cycle disorders. The mechanisms behind most of these disorders are not known in details. In this study we investigated whether the endocrine effect due to SSRI exposure could be detected in well adopted in vitro steroidogenesis assays, two versions of the aromatase enzyme inhibition assay and the H295R cell assay. The five drugs citalopram, fluoxetine, fluvoxamine, paroxetine and sertraline, were shown to inhibit the aromatase enzyme in both types of aromatase assays. The IC50 values ranged from 3 to 600 μM. All five SSRIs, were further investigated in the H295R cell line. All compounds altered the steroid secretion from the cells, the lowest observed effect levels were 0.9 μM and 3.1 μM for sertraline and fluvoxamine, respectively. In general the H295R cell assay was more sensitive to SSRI exposure than the two aromatase assays, up to 20 times more sensitive. This indicates that the H295R cell line is a better tool for screening endocrine disrupting effects. Our findings show that the endocrine effects of SSRIs may, at least in part, be due to interference with the steroidogenesis.

  8. Concordant effects of aromatase inhibitors on gene expression in ER+ Rat and human mammary cancers and modulation of the proteins coded by these genes.

    PubMed

    Lu, Yan; You, Ming; Ghazoui, Zara; Liu, Pengyuan; Vedell, Peter T; Wen, Weidong; Bode, Ann M; Grubbs, Clinton J; Lubet, Ronald A

    2013-11-01

    Aromatase inhibitors are effective in therapy/prevention of estrogen receptor-positive (ER⁺) breast cancers. Rats bearing methylnitrosourea (MNU)-induced ER⁺ mammary cancers were treated with the aromatase inhibitor vorozole (1.25 mg/kg BW/day) for five days. RNA expression showed 162 downregulated and 180 upregulated (P < 0.05 and fold change >1.5) genes. Genes modulated by vorozole were compared with published data from four clinical neoadjuvant trials using aromatase inhibitors (anastrozole or letrozole). More than 30 genes and multiple pathways exhibited synchronous changes in animal and human datasets. Cell-cycle genes related to chromosome condensation in prometaphase [anaphase-prometaphase complex (APC) pathway, including Aurora-A kinase, BUBR1B, TOP2, cyclin A, cyclin B CDC2, and TPX-2)] were downregulated in animal and human studies reflecting the strong antiproliferative effects of aromatase inhibitors. Comparisons of rat arrays with a cell culture study where estrogen was removed from MCF-7 cells showed decreased expression of E2F1-modulated genes as a major altered pathway. Alterations of the cell cycle and E2F-related genes were confirmed in a large independent set of human samples (81 pairs baseline and two weeks anastrozole treatment). Decreases in proliferation-related genes were confirmed at the protein level for cyclin A2, BuRB1, cdc2, Pttg, and TPX-2. Interestingly, the proteins downregulated in tumors were similarly downregulated in vorozole-treated normal rat mammary epithelium. Finally, decreased expression of known estrogen-responsive genes (including TFF, 1,3, progesterone receptor, etc.) were decreased in the animal model. These studies demonstrate that gene expression changes (pathways and individual genes) are similar in humans and the rat model.

  9. Coadministration of the aromatase inhibitor formestane and an isopropanolic extract of black cohosh in a rat model of chemically induced mammary carcinoma.

    PubMed

    Nisslein, Thomas; Freudenstein, Johannes

    2007-04-01

    Non-steroidal as well as steroidal aromatase inhibitors are currently being discussed as alternatives to tamoxifen in the first-line treatment of patients with hormone-dependent breast cancer. Many of these women are in a postmenopausal state and additionally troubled by climacteric complaints. Naturally occurring symptoms like hot flushes and night sweats can be triggered or augmented by anti-hormonal drugs. At the aromatase molecule, steroidal inhibitors like exemestane and formestane compete with the hormonal precursors for the substrate binding site and inactivate the enzyme irreversibly. An isopropanolic extract of the rootstock of black cohosh (iCR), which is a common comedication of aromatase inhibitors in breast cancer patients suffering from climacteric symptoms, contains triterpene glycosides and cinnamic acid esters, both of which possess structural similarities to steroids. We therefore tested a high dose of iCR, guaranteeing an effective uptake of 60 mg herbal substance per kg body weight and shown to influence rat bone and uterus, for putative interactions with two low dosing regimens of 3.5 mg or 5.0 mg formestane per animal and day. We chose a rat model of chemically induced breast cancer and evaluated tumor growth and serum estrogen levels. Compared to a tumor area of 1400 mm2 after 21 days of unopposed tumor growth, formestane treatment, irrespective of concomitant black cohosh application, significantly reduced neoplastic growth by 50%. Formestane also significantly reduced serum estrogen levels, an effect which was also not abolished by iCR. Therefore, in this experimental setting, when challenging two low doses of formestane with a high dose of iCR, our data do not raise concerns against combining aromatase inhibitors with black cohosh.

  10. Cotargeting of CYP-19 (aromatase) and emerging, pivotal signalling pathways in metastatic breast cancer

    PubMed Central

    Daldorff, Stine; Mathiesen, Randi Margit Ruud; Yri, Olav Erich; Ødegård, Hilde Presterud; Geisler, Jürgen

    2017-01-01

    Aromatase inhibition is one of the cornerstones of modern endocrine therapy of oestrogen receptor-positive (ER+) metastatic breast cancer (MBC). The nonsteroidal aromatase inhibitors anastrozole and letrozole, as well as the steroidal aromatase inactivator exemestane, are the preferred drugs and established worldwide in all clinical phases of the disease. However, although many patients suffering from MBC experience an initial stabilisation of their metastatic burden, drug resistance and disease progression occur frequently, following in general only a few months on treatment. Extensive translational research during the past two decades has elucidated the major pathways contributing to endocrine resistance and paved the way for clinical studies investigating the efficacy of novel drug combinations involving aromatase inhibitors and emerging drugable targets like mTOR, PI3K and CDK4/6. The present review summarises the basic research that provided the rationale for new drug combinations involving aromatase inhibitors and the main findings of pivotal clinical trials that have already started to change our way to treat hormone-sensitive MBC. The challenging situation of oestrogen receptor-positive and human epidermal growth factor receptor 2-positive (HER2+) MBC is also shortly reviewed to underline the complexity of the clinical scenario in the heterogeneous subgroups of hormone receptor-positive breast cancer patients and the increasing need for personalised medicine. Finally, we summarise some of the promising findings made with the combination of aromatase inhibitors with other potent endocrine treatment options like fulvestrant, a selective oestrogen receptor downregulator. PMID:27923036

  11. Manipulation of broiler chickens sex differentiation by in ovo injection of aromatase inhibitors, and garlic and tomato extracts.

    PubMed

    Fazli, Nahid; Hassanabadi, Ahmad; Mottaghitalab, Majid; Hajati, Hosna

    2015-11-01

    The influence of in ovo administration of aromatase inhibitors, clomiphen citrate, tomoxifen, and garlic and tomato extracts on sex differentiation in broiler chickens were investigated in 2 experiments. Five hundred, and 1,000 fertile eggs from Ross 308 strain were used in experiments 1 and 2, respectively. In both experiments, eggs were divided into 5 groups: control group (DW, 0.1 mL/egg), tomoxifen (0.05 mg/egg), clomiphene citrate (0.05 mg/egg), garlic and tomato extracts (0.1 mL/egg). Eggs were sanitized and prepared for incubation in a regular automatic hatchery. Experimental preparations were injected into eggs at day 5 of the incubation period. Injection sites on the eggs were cleaned with 70% ethylic alcohol, bored by a needle, and aromatase inhibitors were injected into the white from the thin end of the eggs by insulin syringe and then sealed by melted paraffin. In experiment 1, hatched one-day-old chicks (mixed-sex) were raised till 42 days of age in 25 floor pens with a completely randomized design. Experiment 2 was designed to investigate the effects of sex and treatments on the feed-to-gain ratio of broiler chicks. In experiment 2, hatched one-day-old chicks were feather sexed and raised till 42 days of age in 50 floor pens. A completely randomized design with a 2 × 5 factorial arrangement of treatments (sex×treatment) was used. Gonads of the chicks were checked to determine their sex on day 42 by optic microscope to make sure feather sexing was correct. At the end of both experiments, on day 42, one bird from each pen was slaughtered for carcass analysis. In experiment 1, hatchability and the one-day-old weight of chicks showed no significant differences among treatments (P > 0.05). However, in ovo administration of garlic and tomato extracts caused the highest percentage of male chicks (P < 0.05). Also, the percentage of thighs and wings of the males were significantly higher than those of females (P < 0.05). In experiment 2, feed-to-gain ratio

  12. Breast Cancer, Aromatase Inhibitor Therapy, and Sexual Functioning: A Pilot Study of the Effects of Vaginal Testosterone Therapy

    PubMed Central

    Dahir, Melissa; Travers-Gustafson, Dianne

    2014-01-01

    Introduction Women with breast cancer have better cancer-related outcomes with the use of aromatase inhibitors (AIs), but the physiological suppression of estradiol can negatively affect sexual functioning because of unpleasant urogenital and vaginal symptoms. Local health care practitioners have observed that the benefits of vaginal testosterone in allaying these unpleasant symptoms in women with breast cancer are similar to the benefits of vaginal estrogen in women without breast cancer. Aim The aim of this study was to evaluate the effects of using a daily vaginal testosterone cream on the reported sexual health quality of life in women with breast cancer taking AI therapy. Methods Thirteen postmenopausal women with breast cancer on AI therapy and experiencing symptoms of sexual dysfunction were recruited from an oncology practice. The women were prescribed a 300 μg testosterone vaginal cream daily for 4 weeks. During the first study visit, a vaginal swab was obtained to rule out the presence of Candida species or Gardnerella vaginalis in participants. Women with positive vaginal swabs were treated prior to starting the vaginal testosterone therapy. Main Outcome Measure  The Female Sexual Function Index (FSFI) survey, measuring female sexual health quality of life, was administered during the first study visit and at the final study visit, after completing testosterone therapy. Results Twelve patients completed 4 weeks of daily vaginal testosterone therapy. When compared with baseline FSFI scores, there was a statistically significant improvement for individual domain scores of desire (P = 0.000), arousal (P = 0.002), lubrication (P = 0.018), orgasm (P = 0.005), satisfaction (P = 0.001), and pain (P = 0.000). Total domain scores reflecting sexual health quality of life also improved when compared with baseline (P = 0.000). Conclusions The use of a compounded testosterone vaginal cream applied daily for 4 weeks improves reported sexual health quality of life

  13. Engineering trypsin for inhibitor resistance.

    PubMed

    Batt, Anna R; St Germain, Commodore P; Gokey, Trevor; Guliaev, Anton B; Baird, Teaster

    2015-09-01

    The development of effective protease therapeutics requires that the proteases be more resistant to naturally occurring inhibitors while maintaining catalytic activity. A key step in developing inhibitor resistance is the identification of key residues in protease-inhibitor interaction. Given that majority of the protease therapeutics currently in use are trypsin-fold, trypsin itself serves as an ideal model for studying protease-inhibitor interaction. To test the importance of several trypsin-inhibitor interactions on the prime-side binding interface, we created four trypsin single variants Y39A, Y39F, K60A, and K60V and report biochemical sensitivity against bovine pancreatic trypsin inhibitor (BPTI) and M84R ecotin. All variants retained catalytic activity against small, commercially available peptide substrates [kcat /KM  = (1.2 ± 0.3) × 10(7) M(-1 ) s(-1) . Compared with wild-type, the K60A and K60V variants showed increased sensitivity to BPTI but less sensitivity to ecotin. The Y39A variant was less sensitive to BPTI and ecotin while the Y39F variant was more sensitive to both. The relative binding free energies between BPTI complexes with WT, Y39F, and Y39A were calculated based on 3.5 µs combined explicit solvent molecular dynamics simulations. The BPTI:Y39F complex resulted in the lowest binding energy, while BPTI:Y39A resulted in the highest. Simulations of Y39F revealed increased conformational rearrangement of F39, which allowed formation of a new hydrogen bond between BPTI R17 and H40 of the variant. All together, these data suggest that positions 39 and 60 are key for inhibitor binding to trypsin, and likely more trypsin-fold proteases.

  14. A Pilot Study Comparing the Effect of Flaxseed, Aromatase Inhibitor, and the Combination on Breast Tumor Biomarkers

    PubMed Central

    McCann, Susan E.; Edge, Stephen B.; Hicks, David G.; Thompson, Lilian U.; Morrison, Carl D.; Fetterly, Gerald; Andrews, Christopher; Clark, Kim; Wilton, John; Kulkarni, Swati

    2014-01-01

    Use of complementary approaches is common among breast cancer survivors. Potential interactions between aromatase inhibitors (AI) and high phytoestrogen foods, such as flaxseed (FS) are not often described. We conducted a pilot 2×2 factorial, randomized intervention study between tumor biopsy and resection, in 24 postmenopausal women with estrogen receptor positive (ER+) breast cancer, to assess the effects of flaxseed and anastrozole, and possible interactions between them, on serum steroid hormone and tumor-related characteristics associated with long-term survival (Roswell Park Cancer Institute, 2007–2010). The effect of each treatment vs placebo on outcomes was determined by linear regression adjusting for pre-treatment measure, stage, and grade. Although not statistically significant, mean ERβ expression was approximately 40% lower from pre- to post-intervention in the FS+AI group only. We observed a statistically significant negative association (β±SE −0.3±0.1; p=0.03) for androstenedione in the FS+AI group vs placebo and for DHEA with AI treatment (β±SE −1.6±0.6; p=0.009). Enterolactone excretion was much lower in the FS+AI group compared to the FS group. Our results do not support strong effects of flaxseed on AI activity for selected breast tumor characteristics or serum steroid hormone levels, but suggest AI therapy might reduce the production of circulating mammalian lignans from flaxseed. PMID:24669750

  15. Adverse Drug Event-based Stratification of Tumor Mutations: A Case Study of Breast Cancer Patients Receiving Aromatase Inhibitors.

    PubMed

    Wang, Chen; Zimmermann, Michael T; Prodduturi, Naresh; Chute, Christopher G; Jiang, Guoqian

    2014-01-01

    Adverse drug events (ADEs) are a critical factor for selecting cancer therapy options. The underlying molecular mechanisms of ADEs associated with cancer therapy drugs may overlap with their antineoplastic mechanisms; an aspect of toxicity. In the present study, we develop a novel knowledge-driven approach that provides an ADE-based stratification (ADEStrata) of tumor mutations. We demonstrate clinical utility of the ADEStrata approach through performing a case study of breast invasive carcinoma (BRCA) patients receiving aromatase inhibitors (AI) from The Cancer Genome Atlas (TCGA) (n=212), focusing on the musculoskeletal adverse events (MS-AEs). We prioritized somatic variants in a manner that is guided by MS-AEs codified as 6 Human Phenotype Ontology (HPO) terms. Pathway enrichment and hierarchical clustering of prioritized variants reveals clusters associated with overall survival. We demonstrated that the prediction of per-patient ADE propensity simultaneously identifies high-risk patients experiencing poor outcomes. In conclusion, the ADEStrata approach could produce clinically and biologically meaningful tumor subtypes that are potentially predictive of the drug response to the cancer therapy drugs.

  16. Determination and confirmation of selective estrogen receptor modulators (SERMs), anti-estrogens and aromatase inhibitors in bovine and porcine urine using UHPLC-MS/MS.

    PubMed

    Meijer, Thijs; Essers, Martien L; Kaklamanos, George; Sterk, Saskia S; van Ginkel, Leendert A

    2017-04-01

    Selective estrogen receptor modulators (SERMs), anti-estrogens and aromatase inhibitors are prohibited in human sports doping. However, they also present a risk of being used illegally in animal husbandry for fattening purposes. A method was developed and validated using UHPLC-MS/MS for the determination and confirmation of SERMs, anti-estrogens and aromatase inhibiters in bovine and porcine urine. This method was used in a survey of more than 200 bovine and porcine urine samples from Dutch farms. In 18 out of 103 porcine urine samples (17%) and two out of 114 bovine samples (2%) formestane, an aromatase inhibitor, was detected. None of the other compounds was detected. From human doping control it is known that formestane can, in some cases, be of natural origin. Analyses of reference samples from untreated bovine and porcine animals demonstrated the presence of formestane in bovine animals, but not yet in porcine animals. Future research will focus on whether the detected formestane in porcine and bovine urine is from endogenous or exogenous origin, using GC-c-IRMS.

  17. Synthesis and structure-activity relationship of 1- and 2-substituted-1,2,3-triazole letrozole-based analogues as aromatase inhibitors.

    PubMed

    Doiron, Jérémie; Soultan, Al Haliffa; Richard, Ryan; Touré, Mamadou Mansour; Picot, Nadia; Richard, Rémi; Cuperlović-Culf, Miroslava; Robichaud, Gilles A; Touaibia, Mohamed

    2011-09-01

    A series of bis- and mono-benzonitrile or phenyl analogues of letrozole 1, bearing (1,2,3 and 1,2,5)-triazole or imidazole, were synthesized and screened for their anti-aromatase activities. The unsubstituted 1,2,3-triazole 10a derivative displayed inhibitory activity comparable with that of the aromatase inhibitor, letrozole 1. Compound 10a, bearing a 1,2,3-triazole, is also 10000-times more tightly binding than the corresponding analogue 25 bearing a 1,2,5-triazole, which confirms the importance of a nitrogen atom at position 3 or 4 of the 5-membered ring needed for high activity. The effect on human epithelial adrenocortical carcinoma cell line (H295R) proliferation was also evaluated. The compound 10j (IC(50) = 4.64 μM), a letrozole 1 analogue bearing para-cyanophenoxymethylene-1,2,3-triazole decreased proliferation rates of H295R cells by 76 and 99% in 24 and 72 h respectively. Computer calculations, using quantum ab initio structures, suggest a possible correlation between anti-aromatase activity and the distance between the nitrogen in position 3 or 4 of triazole nitrogen and the cyano group nitrogen.

  18. Persistent endocrine disruption effects in medaka fish with early life-stage exposure to a triazole-containing aromatase inhibitor (letrozole).

    PubMed

    Liao, Pei-Han; Chu, Szu-Hung; Tu, Tzu-Yi; Wang, Xiao-Huan; Lin, Angela Yu-Chen; Chen, Pei-Jen

    2014-07-30

    Letrozole (LET) is a triazole-containing drug that can inhibit the activity of cytochrome P450 aromatase. It is an environmentally emerging pollutant because of its broad use in medicine and frequent occurrence in aquifers receiving the effluent of municipal or hospital wastewater. However, the toxic impact of LET on fish populations remains unclear. We exposed medaka fish (Oryzias latipes) at an early stage of sexual development to a continuous chronic LET at environmentally relevant concentrations and assessed the endocrine disruption effects in adulthood and the next generation. LET exposure at an early life stage persistently altered phenotypic sex development and reproduction in adults and skewed the sex ratio in progeny. As well, LET exposure led to a gender-different endocrine disruption as seen by the interruption in gene expression responsible for estrogen synthesis and metabolism and fish reproduction. LET interfering with the aromatase system in early life stages of medaka can disrupt hormone homeostasis and reproduction. This potent aromatase inhibitor has potential ecotoxicological impact on fish populations in aquatic environments.

  19. Phase II study of glucosamine with chondroitin on aromatase inhibitor-associated joint symptoms in women with breast cancer

    PubMed Central

    Greenlee, Heather; Crew, Katherine D.; Shao, Theresa; Kranwinkel, Grace; Kalinsky, Kevin; Maurer, Matthew; Brafman, Lois; Insel, Beverly; Tsai, Wei Yann

    2013-01-01

    Purpose Many women with hormone receptor-positive breast cancer discontinue effective aromatase inhibitor (AI) treatment due to joint symptoms. Methods We conducted a single-arm, open-label, phase II study evaluating glucosamine-sulfate (1,500 mg/day)+ chondroitin-sulfate (1,200 mg/day) for 24 weeks to treat joint pain/stiffness in postmenopausal women with early stage breast cancer who developed moderate-to-severe joint pain after initiating AIs. The primary endpoint was improvement in pain/stiffness at week 24 assessed by the Outcome Measure in Rheumatology Clinical Trials and Osteoarthritis Research Society International (OMERACT-OARSI) criteria. Secondary endpoints assessed changes in pain, stiffness, and function using the Western Ontario and McMaster Universities Osteoarthritis (WOMAC) Index for hips/knees and the Modified Score for the Assessment and Quantification of Chronic Rheumatoid Affections of the Hands (M-SACRAH) for hands/wrists. The Brief Pain Inventory (BPI) assessed pain interference, severity, and worst pain. Results Of 53 patients enrolled, 39 were evaluable at week 24. From baseline to week 24, 46 % of patients improved according to OMERACT-OARSI criteria. At week 24, there were improvements (all P<0.05) in pain and function as assessed by WOMAC and M-SACRAH, and in pain interference, severity, and worst pain as assessed by BPI. Estradiol levels did not change from baseline. The most commonly reported side effects were headache (28 %), dyspepsia (15 %), and nausea (17 %). Conclusions In this single-arm study, 24 weeks of glucosamine/chondroitin resulted in moderate improvements in AI-induced arthralgias, with minimal side effects, and no changes in estradiol levels. These results suggest a need to evaluate efficacy in a placebo-controlled trial. PMID:23111941

  20. Effects of the aromatase inhibitor fadrozole on plasma sex steroid secretion, spermatogenesis and epididymis morphology in the lizard, Podarcis sicula.

    PubMed

    Cardone, Anna; Comitato, Raffaella; Bellini, Luigi; Angelini, Francesco

    2002-09-01

    Recently, increasing importance has been attached to the role of estrogens and their receptors in male reproduction, since they have been found to be abundant in the male reproductive tract. In the lizard, Podarcis sicula, a seasonal breeder, estrogens seem to be involved in the regulation of testicular activity. Particularly, it has been hypothesized that the block of spermatogenesis and the complete regression of the epididymis and other secondary sexual characters (SSCs) in autumn might be due to high estrogen levels. To investigate the role of estrogens in the reproductive process of male lizards, we utilized Fadrozole ((AI) [4-(5,6,7,8-tetrahydroimidazole [1,5-a] pyridin-5-yl)-benzonitrile monohydrochloride] (CGS 16949A)), a nonsteroidal inhibitor of aromatase, the enzyme involved in the aromatization of androgens to estrogens, evaluating its effects on plasma sex-hormone release, spermatogenesis and epididymis morphology. For this purpose, adult male lizards, captured during the autumnal recrudescence, were intraperitoneally injected with 0.5 microg and 5 microg/g/body weight of AI for 15 and 30 days. In the animals treated with the higher AI dose, estrogen levels decreased if compared to the control groups, whereas androgen levels increased. Furthermore, histologic sections of testes and epididymes showed that the 30-day treatment with AI-induced spermatogenesis resumption with release of sperms into the large lumen of the seminiferous tubules, and the epididymes appeared more developed with moderately secreting columnar canal cells. Therefore, it is proposed that failure of spermatogenesis in autumn might be due to high estrogen levels.

  1. Targeted Metabolomics Approach To Detect the Misuse of Steroidal Aromatase Inhibitors in Equine Sports by Biomarker Profiling.

    PubMed

    Chan, George Ho Man; Ho, Emmie Ngai Man; Leung, David Kwan Kon; Wong, Kin Sing; Wan, Terence See Ming

    2016-01-05

    The use of anabolic androgenic steroids (AAS) is prohibited in both human and equine sports. The conventional approach in doping control testing for AAS (as well as other prohibited substances) is accomplished by the direct detection of target AAS or their characteristic metabolites in biological samples using hyphenated techniques such as gas chromatography or liquid chromatography coupled with mass spectrometry. Such an approach, however, falls short when dealing with unknown designer steroids where reference materials and their pharmacokinetics are not available. In addition, AASs with fast elimination times render the direct detection approach ineffective as the detection window is short. A targeted metabolomics approach is a plausible alternative to the conventional direct detection approach for controlling the misuse of AAS in sports. Because the administration of AAS of the same class may trigger similar physiological responses or effects in the body, it may be possible to detect such administrations by monitoring changes in the endogenous steroidal expression profile. This study attempts to evaluate the viability of using the targeted metabolomics approach to detect the administration of steroidal aromatase inhibitors, namely androst-4-ene-3,6,17-trione (6-OXO) and androsta-1,4,6-triene-3,17-dione (ATD), in horses. Total (free and conjugated) urinary concentrations of 31 endogenous steroids were determined by gas chromatography-tandem mass spectrometry for a group of 2 resting and 2 in-training thoroughbred geldings treated with either 6-OXO or ATD. Similar data were also obtained from a control (untreated) group of in-training thoroughbred geldings (n = 28). Statistical processing and chemometric procedures using principle component analysis and orthogonal projection to latent structures-discriminant analysis (OPLS-DA) have highlighted 7 potential biomarkers that could be used to differentiate urine samples obtained from the control and the treated groups

  2. Metabolomic, behavioral, and reproductive effects of the aromatase inhibitor fadrozole hydrochloride on the unionid mussel Lampsilis fasciola.

    PubMed

    Leonard, Jeremy A; Cope, W Gregory; Barnhart, M Christopher; Bringolf, Robert B

    2014-09-15

    Androgen-induced masculinization of female aquatic biota poses concerns for natural population stability. This research evaluated the effects of a twelve day exposure of fadrozole hydrochloride on the metabolism and reproductive status of the unionid mussel Lampsilis fasciola. Although this compound is not considered to be widespread in the aquatic environment, it was selected as a model aromatase (enzyme that converts testosterone to estradiol) inhibitor. Adult mussels were exposed to a control and 3 concentrations of fadrozole (2μg/L, 20μg/L, and 50μg/L), and samples of gill tissue were taken on days 4 and 12 for metabolomics analysis. Gills were used because of the variety of critical processes they mediate, such as feeding, ion exchange, and siphoning. Daily observed mussel behavior included female mantle display, foot protrusion, siphoning, and larval (glochidia) releases. Glochidia mortality was significantly higher in the 20μg/L treatment. Fewer conglutinate (packets of glochidia) releases were observed in the 50μg/L treatment, and mortality was highly correlated to release numbers. Foot protrusion was significantly higher in females in nearly all treatments, including the control, during the first 4days of observations. However, this sex difference was observed only in the 50μg/L treatment during the last 8days. Generally, metabolites were significantly altered in female gill tissue in the 2μg/L treatment whereas males were mostly affected only at the highest (50μg/L) treatment. Both sexes also revealed significant reductions in fadrozole-induced metabolic effects in gill tissue sampled after 12days compared to tissue sampled after 4days, indicating time-dependent mechanisms of disruptions in metabolic pathways and homeostatic processes to compensate for such disruptions.

  3. Kinetics, prognostic and predictive values of ESR1 circulating mutations in metastatic breast cancer patients progressing on aromatase inhibitor

    PubMed Central

    Clatot, Florian; Perdrix, Anne; Augusto, Laetitia; Beaussire, Ludivine; Delacour, Julien; Calbrix, Céline; Sefrioui, David; Viailly, Pierre-Julien; Bubenheim, Michael; Moldovan, Cristian; Alexandru, Cristina; Tennevet, Isabelle; Rigal, Olivier; Guillemet, Cécile; Leheurteur, Marianne; Gouérant, Sophie; Petrau, Camille; Théry, Jean-Christophe; Picquenot, Jean-Michel; Veyret, Corinne; Frébourg, Thierry; Jardin, Fabrice

    2016-01-01

    Purpose To assess the prognostic and predictive value of circulating ESR1 mutation and its kinetics before and after progression on aromatase inhibitor (AI) treatment. Patients and methods ESR1 circulating D538G and Y537S/N/C mutations were retrospectively analyzed by digital droplet PCR after first-line AI failure in patients treated consecutively from 2010 to 2012 for hormone receptor-positive metastatic breast cancer. Progression-free survival (PFS) and overall survival (OS) were analyzed according to circulating mutational status and subsequent lines of treatment. The kinetics of ESR1 mutation before (3 and 6 months) and after (3 months) AI progression were determined in the available archive plasmas. Results Circulating ESR1 mutations were found at AI progression in 44/144 patients included (30.6%). Median follow-up from AI initiation was 40 months (range 4-94). The median OS was decreased in patients with circulating ESR1 mutation than in patients without mutation (15.5 versus 23.8 months, P=0.0006). The median PFS was also significantly decreased in patients with ESR1 mutation than in patients without mutation (5.9 vs 7 months, P=0.002). After AI failure, there was no difference in outcome for patients receiving chemotherapy (n = 58) versus non-AI endocrine therapy (n=51) in patients with and without ESR1 mutation. ESR1 circulating mutations were detectable in 75% of all cases before AI progression, whereas the kinetics 3 months after progression did not correlate with outcome. Conclusion ESR1 circulating mutations are independent risk factors for poor outcome after AI failure, and are frequently detectable before clinical progression. Interventional studies based on ESR1 circulating status are warranted. PMID:27801670

  4. Aromatase inhibitors associated musculoskeletal disorders and bone fractures in postmenopausal breast cancer patients: a result from Chinese population.

    PubMed

    Xu, Lu; Wang, Jue; Xue, Dan-Dan; He, Wei

    2014-09-01

    As the prognosis of early breast cancer patients improves, the long-term safety of aromatase inhibitors (AIs) is increasingly important. In the present study, we retrospectively investigated the incidences of musculoskeletal disorders (MSDs) and bone fractures in a cohort of Chinese postmenopausal patients with breast cancer. Data of postmenopausal patients with breast cancer were collected. Among which, 70 patients received AIs therapy (median follow-up of 32.5 months), 52 patients received tamoxifen (TAM), and 89 patients received no endocrine therapy (NE). Baseline characteristics, incidence of MSDs and bone fractures were analyzed and compared. When compared with NE group (40.4 %, 36/89), more patients in AIs group developed MSDs (72.9 %, 51/70, adjusted odds ratio (AOR) = 3.30, 95 % confidence interval (CI) = 1.59-6.88, P = 0.001). But no difference was found between TAM group (36.5 %, 19/52, AOR = 0.70, 95 % CI = 0.32-1.52, P = 0.372) and NE group. About 39.7 months after initial AIs therapy, nine patients in AI group developed bone fractures in different sites, and the bone fracture rate was significantly increased (12.9 %, 9/70, adjusted hazard ratio (AHR) = 20.08, 95 % CI = 1.72-234.08, P = 0.017) in comparison with NE group (1.1 %, 1/89). Moreover, the bone fracture rate of TAM group was not different from NE group (1.9 %, 1/52, AHR = 2.64, 95 % CI = 0.14-48.73, P = 0.513). AIs therapy may induce increased rates of MSDs and bone fractures in Chinese population of postmenopausal breast cancer patients, whereas TAM therapy did not help reduce the incidences of MSDs and bone fractures.

  5. Induction of CYP1A and cyp2-mediated arachidonic acid epoxygenation and suppression of 20-hydroxyeicosatetraenoic acid by imidazole derivatives including the aromatase inhibitor vorozole.

    PubMed

    Diani-Moore, Silvia; Papachristou, Fotini; Labitzke, Erin; Rifkind, Arleen B

    2006-08-01

    Cytochrome P450 (P450) enzymes metabolize the membrane lipid arachidonic acid to stable biologically active epoxides [eicosatrienoic acids (EETs)] and 20-hydroxyeicosatetraenoic acid (20-HETE). These products have cardiovascular activity, primarily acting as vasodilators and vasoconstrictors, respectively. EET formation can be increased by the prototype CYP1A or CYP2 inducers, 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) or phenobarbital (PB), respectively. We report here that imidazole derivative drugs: the anthelminthics, albendazole and thiabendazole; the proton pump inhibitor, omeprazole; the thromboxane synthase inhibitor, benzylimidazole; and the aromatase (CYP19) inhibitor vorozole (R76713, racemate; and R83842, (+) enantiomer) increased hepatic microsomal EET formation in a chick embryo model. Albendazole increased EETs by transcriptional induction of CYP1A5 and the others by combined induction of CYP1A5 and CYP2H, the avian orthologs of mammalian CYP1A2 and CYP2B, respectively. All inducers increased formation of the four EET regioisomers, but TCDD and albendazole had preference for 5,6-EET and PB and omeprazole for 14,15-EET. Vorozole, benzylimidazole, and TCDD also suppressed 20-HETE formation. Vorozole was a remarkably effective and potent inducer of multiple hepatic P450s at a dose range which overlapped its inhibition of ovarian aromatase. Increased CYP1A activity in mouse Hepa 1-6 and human HepG2 cells by vorozole and other imidazole derivatives demonstrated applicability of the findings to mammalian cells. The findings suggest that changes in P450-dependent arachidonic acid metabolism may be a new source of side effects for drugs that induce CYP1A or CYP2. They demonstrate further that in vivo induction of multiple hepatic P450s produces additive increases in arachidonic acid epoxygenase activity and can occur concurrently with inhibition of ovarian aromatase activity.

  6. [Combination of trastuzumab, aromatase inhibitor and anti-cancer drugs obtained a good prognosis for an inoperable stage III B breast cancer patient with giant skin ulceration].

    PubMed

    Takeda, Yasutaka; Tanaka, Noriyoshi; Konishi, Juichiro

    2012-04-01

    A 68-year-old woman who had an inoperable, ER-positive, PgR-positive and HER2-positive advanced breast cancer with giant skin ulceration has been treated with the combination of trastuzumab, aromatase inhibitor and anti-cancer drugs. She was thus well-controll for over 9 years. Trastuzumab was administered more than 400 times, but no cardiac toxicity has been observed. The synergistic efficacy of the combination of trastuzumab and anti-cancer drugs was already proven, but it has recently been reported that concurrent treatment of trastuzumab and endocrine therapy improves the prognoses of triple positive breast cancer patients.

  7. Natural compounds with aromatase inhibitory activity: an update.

    PubMed

    Balunas, Marcy J; Kinghorn, A Douglas

    2010-08-01

    Several synthetic aromatase inhibitors are currently in clinical use for the treatment of postmenopausal women with hormone-receptor positive breast cancer. However, these treatments may lead to untoward side effects and so the search for new aromatase inhibitors continues, especially those for which the activity is promoter-specific, targeting the breast-specific promoters I.3 and II. Recently, numerous natural compounds have been found to inhibit aromatase in noncellular, cellular, and IN VIVO studies. These investigations, covering the last two years, as well as additional studies that have focused on the evaluation of natural compounds as promoter-specific aromatase inhibitors or as aromatase inducers, are described in this review.

  8. Modulation of Aromatase by Phytoestrogens

    PubMed Central

    Lephart, Edwin D.

    2015-01-01

    The aromatase enzyme catalyzes the conversion of androgens to estrogens in many human tissues. Estrogens are known to stimulate cellular proliferation associated with certain cancers and protect against adverse symptoms during the peri- and postmenopausal intervals. Phytoestrogens are a group of plant derived naturally occurring compounds that have chemical structures similar to estrogen. Since phytoestrogens are known to be constituents of animal/human food sources, these compounds have received increased research attention. Phytoestrogens may contribute to decreased cancer risk by the inhibition of aromatase enzyme activity and CYP19 gene expression in human tissues. This review covers (a) the aromatase enzyme (historical descriptions on function, activity, and gene characteristics), (b) phytoestrogens in their classifications and applications to human health, and (c) a chronological coverage of aromatase activity modulated by phytoestrogens from the early 1980s to 2015. In general, phytoestrogens act as aromatase inhibitors by (a) decreasing aromatase gene expression, (b) inhibiting the aromatase enzyme itself, or (c) in some cases acting at both levels of regulation. The findings presented herein are consistent with estrogen's impact on health and phytoestrogen's potential as anticancer treatments, but well-controlled, large-scale studies are warranted to determine the effectiveness of phytoestrogens on breast cancer and age-related diseases. PMID:26798508

  9. Anti-aromatase chemicals in red wine.

    PubMed

    Eng, E T; Williams, D; Mandava, U; Kirma, N; Tekmal, R R; Chen, S

    2002-06-01

    Estrogen synthesized in situ plays a more important role in breast cancer cell proliferation than does circulating estrogen. Aromatase is the enzyme that converts androgen to estrogen and is expressed at a higher level in breast cancer tissue than in surrounding noncancer tissue. A promising route of chemoprevention against breast cancer may be through the suppression of in situ estrogen formation using aromatase inhibitors. A diet high in fruits and vegetables may reduce the incidence of breast cancer, because they contain phytochemicals that can act as aromatase inhibitors. In our previous studies, we found that grapes and wine contain potent phytochemicals that can inhibit aromatase. We show that red wine was more effective than white wine in suppressing aromatase activity. Interestingly, our results from white wine studies suggest a weak inductive effect of alcohol on aromatase activity. On the other hand, the potent effect of anti-aromatase chemicals in red wine overcomes the weak inductive effect of alcohol in wine. Several purification procedures were performed on whole red wine to separate active aromatase inhibitors from non-active compounds. These techniques included liquid-liquid extraction, silica gel chromatography, various solid phase extraction (SPE) columns, and high performance liquid chromatography. An active Pinot Noir red wine SPE C18 column fraction (20% acetonitrile:water) was more effective than complete Pinot Noir wine in suppressing aromatase assay. This red wine extract was further analyzed in a transgenic mouse model in which aromatase was over-expressed in mammary tissue. Our gavaged red wine extract completely abrogated aromatase-induced hyperplasia and other neoplastic changes in mammary tissue. These results suggest that red wine or red wine extract may be a chemopreventive diet supplement for postmenopausal women who have a high risk of breast cancer. Further research is underway to purify and characterize the active compounds in red

  10. Biological aromatization of delta4,6- and delta1,4,6-androgens and their 6-alkyl analogs, potent inhibitors of aromatase.

    PubMed

    Numazawa, M; Yoshimura, A

    1999-01-01

    Enzymic aromatization of delta6- and delta1,6-derivatives of the natural substrate androstenedione with human placental aromatase was first studied using gas-chromatography-mass spectrometry. The two steroids were aromatized with apparent Km and Vmax values of 62 nM and 32 pmol/min/mg protein for the delta6-steroid and 167 nM and 10 pmol/min/mg protein for the delta1,6-steroid, respectively. We next explored the aromatization of a series of 6-alkyl (methyl, ethyl, n-propyl, and n-pentyl)-substituted delta6-androstenediones and their delta1,6-analogs, potent competitive inhibitors of aromatase, to gain insight into the relationships between the inhibitory activity of the 6-alkyl-C19 steroids and their ability to serve as a substrate of aromatase. In a series of the delta1,6-androstenediones, all the 6-alkyl steroids were more efficient substrates than the parent delta1,6-steroid in which the aromatization rates of the alkyl steroids were about 2-fold that of the parent steroid, in contrast, all of the 6-alkyl-substituted delta6-androstenediones were converted into the corresponding 6-alkyl-delta6-estrogens with the rates of less than about a half that of the parent steroid. These results indicate that the 6-alkyl function decreases the aromatization rate of the delta6-steroid but enhances that of the delta1,6-steroid. The relative apparent Km values for the C19 steroids obtained in this study are different from the relative Ki values obtained previously, indicating that a good inhibitor is not essentially a good substrate in the 6-alkyl-substituted delta6- and delta1,6-androstenedione series.

  11. Effects of the traditional Chinese medicine Yi Shen Jian Gu granules on aromatase inhibitor-associated musculoskeletal symptoms: a study protocol for a multicenter, randomized, controlled clinical trial

    PubMed Central

    2014-01-01

    Background Aromatase inhibitors (AIs) are widely used as an adjuvant endocrine treatment in postmenopausal women with early-stage breast cancer. One of the main adverse effects of AIs is musculoskeletal symptoms, which leads to a lower quality of life and poor adherence to AI treatment. To date, no effective management of aromatase inhibitor-associated musculoskeletal symptoms (AIMSS) has been developed. Methods/design To determine whether the traditional Chinese medicine Yi Shen Jian Gu granules could effectively manage AIMSS we will conduct a multicenter, randomized, double-blind, placebo-controlled clinical trial. Patients experiencing musculoskeletal symptoms after taking AIs will be enrolled and treated with traditional Chinese medicine or placebo for 12 weeks. The primary outcome measures include Brief Pain Inventory-Short Form, Western Ontario and McMaster Universities Osteoarthritis Index, and Modified Score for the Assessment and Quantification of Chronic Rheumatoid Affections of the Hands, which will be obtained at baseline and at 4, 8, 12 and 24 weeks. Discussion The results of this study will provide a new strategy to help relieve AIMSS. Trial registration ISCTN: ISRCTN06129599 (assigned 14 August 2013). PMID:24885324

  12. Aromatase Inhibition in a Transcriptional Network Context

    EPA Science Inventory

    A variety of chemicals in the environment have the potential to inhibit aromatase, an enzyme critical to estrogen synthesis. We examined the responses of female fathead minnow ovaries (FHM, Pimephales promelas) to a model aromatase inhibitor, fadrozole, using a transcriptional ne...

  13. Inhibition of peripheral aromatization in baboons by an enzyme-activated aromatase inhibitor (MDL 18,962)

    SciTech Connect

    Longcope, C.; Femino, A.; Johnston, J.O.

    1988-05-01

    The peripheral aromatization ((rho)BM) of androstenedione (A) and testosterone (T) was measured before and after administration of the aromatase inhibitor 10-(2 propynyl)estr-4-ene-3,17-dione (MDL-18,962) to five mature female baboons, Papio annubis. The measurements were made by infusing (3H)androstenedione/(14C)estrone or (3H)testosterone/(14C)estradiol for 3.5 h and collecting blood samples during the infusions and all urine for 96 h from the start of the infusion. Blood samples were analyzed for radioactivity as infused and product steroids, and the data were used to calculate MCRs. An aliquot of the pooled urine was analyzed for the glucuronides of estrone and estradiol and used to calculate the (rho)BM. MDL-18,962 was administered as a pulse in polyethylene glycol-400 (1-5 ml) either iv or via gastric tube 30 min before administration of the radiolabeled steroids. Control studies were done with and without polyethylene glycol-400 administration. When MDL-18,962 was given iv at 4 mg/kg, the aromatization of A was decreased 91.8 +/- 0.9% from the control value of 1.23 +/- 0.13% to 0.11 +/- 0.01%. At the same dose, aromatization of T was decreased 82.0 +/- 7.1%, from a control value of 0.20 +/- 0.03% to 0.037 +/- 0.018%. When MDL-18,962 was given iv at doses of 0.4, 0.1, 0.04, and 0.01 mg/kg, the values for aromatization of A were 0.16 +/- 0.03%, 0.18 +/- 0.06%, 0.37 +/- 11%, and 0.65 +/- 0.09%, respectively. The administration of MDL-18,962 via gastric tube at 4 mg/kg as a pulse decreased the aromatization of A from 1.35 +/- 0.06% to 0.43 +/- 0.12%, an inhibition of 67.2 +/- 10.7%. When administered via gastric tube daily for 5 days at 4 mg/kg, the aromatization of A fell from 1.35 +/- 0.06% to 0.063 +/- 0.003%, an inhibition of 84.4 +/- 0.5%.

  14. Preventive effect of zoledronic acid on aromatase inhibitor-associated bone loss for postmenopausal breast cancer patients receiving adjuvant letrozole

    PubMed Central

    Sun, Shengliang; Wang, Fuchao; Dou, Honglei; Zhang, Longqiang; Li, Jiwen

    2016-01-01

    Background This study aims to compare the efficacy and safety between zoledronic acid combined with calcium and calcium alone to prevent aromatase inhibitor-associated bone loss for postmenopausal breast cancer patients receiving adjuvant letrozole. Methods One hundred twenty patients were randomly divided into two groups, A and B. Patients in group A (n=60) received modified radical mastectomy or breast-conserving surgery + four cycles of AC followed by T regimen (optional) + radiotherapy (optional) + letrozole 2.5 mg daily + calcium 500 mg twice daily + vitamin D 400 international units daily +4 mg of zoledronic acid every 6 months, while patients in group B (n=60) were not given zoledronic acid and the rest of the treatments of group B were the same as group A. All the patients were followed up for 1 year. The primary endpoint was the intrapatient percentage change in lumbar spine (LS) bone mineral density (BMD) from baseline to month 12. Secondary endpoints included the percentage change in total hip (TH) and femoral neck (FN) BMD, the incidence of osteoporosis, the incidence of a clinically meaningful 5% decline in BMD at 1 year, change of serum N-telopeptide of type 1 collagen (NTX) and bone-specific alkaline phosphatase (BSAP) concentrations. Results Patients in group A had a statistically significant higher average change and average percent change in LS, FN, and TH than group B. Group A had a statistically significant lower incidence of a clinically meaningful loss of bone density at the LS, FN, or TH than Group B. The incidence of osteoporosis in group A was significantly lower than group B. The decreases in NTX and BSAP concentrations from baseline to month 12 in patients of group A were significant; in contrast, patients in group B were found to have increases in NTX and BSAP concentrations from baseline. The most common adverse reactions in patients are flu-like symptoms (38%), bone pain (28%), and joint pain (20%). Conclusion AI-associated bone loss

  15. Resistance to AHAS inhibitor herbicides: current understanding.

    PubMed

    Yu, Qin; Powles, Stephen B

    2014-09-01

    Acetohydroxyacid synthase (AHAS) inhibitor herbicides currently comprise the largest site-of-action group (with 54 active ingredients across five chemical groups) and have been widely used in world agriculture since they were first introduced in 1982. Resistance evolution in weeds to AHAS inhibitors has been rapid and identified in populations of many weed species. Often, evolved resistance is associated with point mutations in the target AHAS gene; however non-target-site enhanced herbicide metabolism occurs as well. Many AHAS gene resistance mutations can occur and be rapidly enriched owing to a high initial resistance gene frequency, simple and dominant genetic inheritance and lack of major fitness cost of the resistance alleles. Major advances in the elucidation of the crystal structure of the AHAS (Arabidopsis thaliana) catalytic subunit in complex with various AHAS inhibitor herbicides have greatly improved current understanding of the detailed molecular interactions between AHAS, cofactors and herbicides. Compared with target-site resistance, non-target-site resistance to AHAS inhibitor herbicides is less studied and hence less understood. In a few well-studied cases, non-target-site resistance is due to enhanced rates of herbicide metabolism (metabolic resistance), mimicking that occurring in tolerant crop species and often involving cytochrome P450 monooxygenases. However, the specific herbicide-metabolising, resistance-endowing genes are yet to be identified in resistant weed species. The current state of mechanistic understanding of AHAS inhibitor herbicide resistance is reviewed, and outstanding research issues are outlined.

  16. Depsidones, aromatase inhibitors and radical scavenging agents from the marine-derived fungus Aspergillus unguis CRI282-03.

    PubMed

    Sureram, Sanya; Wiyakrutta, Suthep; Ngamrojanavanich, Nattaya; Mahidol, Chulabhorn; Ruchirawat, Somsak; Kittakoop, Prasat

    2012-04-01

    Three new depsidones ( 1, 3, and 4), a new diaryl ether ( 5), and a new natural pyrone ( 9) (synthetically known), together with three known depsidones, nidulin ( 6), nornidulin ( 7), and 2-chlorounguinol ( 8), were isolated from the marine-derived fungus ASPERGILLUS UNGUIS CRI282-03. Aspergillusidone C ( 4) showed the most potent aromatase inhibitory activity with the IC (50) value of 0.74 µM, while depsidones 1, 3, 6- 8 inhibited aromatase with IC (50) values of 1.2-11.2 µM. It was found that the structural feature of depsidones, not their corresponding diaryl ether derivatives (e.g. 5), was important for aromatase inhibitory activity. Aspergillusidones A ( 1) and B ( 3) showed radical scavenging activity in the XXO assay with IC (50) values of 16.0 and < 15.6 µM, respectively. Compounds 1 and 3- 7 were mostly inactive or showed only weak cytotoxic activity against HuCCA-1, HepG2, A549, and MOLT-3 cancer cell lines.

  17. Novel aromatase inhibitors selection using induced fit docking and extra precision methods: Potential clinical use in ER-alpha-positive breast cancer

    PubMed Central

    Kumavath, Ranjith; Azad, Manan; Devarapalli, Pratap; Tiwari, Sandeep; Kar, Shreya; Barh, Debmalya; Azevedo, Vasco; Kumar, Alan Prem

    2016-01-01

    Aromatase (CYP19A1) the key enzyme of estrogen biosynthesis, is often deregulated in breast cancer patients. It catalyzes the conversion of androgen to estrogen, thus responsible for production of estrogen in human body. However, it causes over-production of estrogen which eventually leads to proliferation of breast cancer cells. Identification of new small molecule inhibitors targeted against CYP19A1 therefore, facilitates to increase drug sensitivity of cancer cells. In this scenario, the present study aims to identify new molecules which could block or suppress the activity of aromatase enzyme by molecular docking studies using Schrödinger-Maestro v9.3. In this study we used in silico approach by modeling CYP19A1 protein the strcture was subjected to protein preparation wizard; to add hydrogen and optimize the protonation states of Thr310 and Ser478 and Asp309 residues. Active site of the CYP19A1 protein was identified using SiteMap tool of Scchrodinger package. We further carried out docking studies by means of Glid, with various ligands. Based on glid score, potential ligands were screeened and their interaction with CYP19A1 was identified. The best hits were further screened for Lipinski’s rule for drug-likeliness and bioactivity scoring properties. Thus, we report two rubivivaxin and rhodethrin compounds that have successfully satisfied all in silico parameters, necessitating further in vitro and in vivo studies. PMID:28293075

  18. In vivo visualization of aromatase in animals and humans

    PubMed Central

    Biegon, Anat

    2015-01-01

    Aromatase catalyzes the last and obligatory step in the biosynthesis of estrogens across species. In vivo visualization of aromatase can be performed using positron emission tomography (PET) with radiolabeled aromatase inhibitors such as [11C]vorozole. PET studies in rats, monkeys and healthy human subjects demonstrate widespread but heterogeneous aromatase availability in brain and body, which appears to be regulated in a species, sex and region-specific manner. Thus, aromatase availability is high in brain amygdala and in ovaries of all species examined to date, with males demonstrating higher levels than females in all comparable organs. However, the highest concentrations of aromatase in the human brain are found in specific nuclei of the thalamus while the highest levels in rats and monkeys are found in the amygdala. Regional brain aromatase availability is increased by androgens and inhibited by nicotine. Future studies may improve diagnosis and treatment in brain disorders and cancers overexpressing aromatase. PMID:26456904

  19. Synthesis of 6- or 4-functionalized indoles via a reductive cyclization approach and evaluation as aromatase inhibitors.

    PubMed

    Lézé, Marie-Pierre; Palusczak, Anja; Hartmann, Rolf W; Le Borgne, Marc

    2008-08-15

    Two new series of benzonitrile derivatives on position 6 or 4 of indole ring were successfully synthesized via a Leimgruber-Batcho reaction. All the compounds were evaluated in vitro on the inhibition of aromatase (CYP19) and 17alpha-hydroxylase-C17,20-lyase (CYP17). The racemate, 4-[(1H-imidazol-1-yl)(1H-indol-4-yl)methyl]benzonitrile 9, showed high level of inhibitory activity towards CYP19 (IC(50)=11.5 nM).

  20. ETAR antagonist ZD4054 exhibits additive effects with aromatase inhibitors and fulvestrant in breast cancer therapy, and improves in vivo efficacy of anastrozole.

    PubMed

    Smollich, Martin; Götte, Martin; Fischgräbe, Jeanett; Macedo, Luciana F; Brodie, Angela; Chen, Shiuan; Radke, Isabel; Kiesel, Ludwig; Wülfing, Pia

    2010-09-01

    Endothelin-1 (ET-1) and endothelin A receptor (ETAR) contribute to the development and progression of breast carcinomas by modulating cell proliferation, angiogenesis, and anti-apoptosis. We investigated antitumoral effects of the specific ETAR antagonist ZD4054 in breast cancer cells and xenografts, and assessed antitumoral efficacy of the combinations of ZD4054 with aromatase inhibitors and fulvestrant. Gene expression changes were assessed by quantitative real-time PCR. Cell proliferation was measured using alamarBlue; migration and invasion assays were performed using modified Boyden chambers. Evaluating the antitumoral efficacy of ZD4054 in vivo, different breast cancer models were employed using nude mice xenografts. ZD4054 reduced ET-1 and ETAR expression in MCF-7, MDA-MB-231, and MDA-MB-468 breast cancer cells in a concentration-dependent manner. ZD4054 inhibited invasion by up to 37.1% (P = 0.022). Combinations of ZD4054 with either anastrozole or letrozole produced significant reductions in migration of aromatase-overexpressing MCF-7aro cells (P < 0.05). Combination of ZD4054 with fulvestrant reduced MCF-7 cell migration and invasion by 36.0% (P = 0.027) and 56.7% (P < 0.001), respectively, with effects significantly exceeding those seen with either compound alone. Regarding tumor volume reduction in vivo, ZD4054 (10 mg/kg) was equipotent to fulvestrant (200 mg/kg) and exhibited additive effects with anastrozole (0.5 mg/kg). These data are the first indicating that selective ETAR antagonism by ZD4054 displays antitumoral activity on breast cancer cells in vitro and in vivo. Our data strongly support a rationale for the clinical use of ZD4054 in combination with endocrine therapies.

  1. Aromatase inhibitors with or without luteinizing hormone-releasing hormone agonist for metastatic male breast cancer: report of four cases and review of the literature.

    PubMed

    Kuba, Sayaka; Ishida, Mayumi; Oikawa, Masahiro; Nakamura, Yoshiaki; Yamanouchi, Kosho; Tokunaga, Eriko; Taguchi, Kenichi; Esaki, Taito; Eguchi, Susumu; Ohno, Shinji

    2016-11-01

    The roles of aromatase inhibitors (AIs) and luteinizing hormone-releasing hormone (LH-RH) agonists in the management of male breast cancer remain uncertain, with no reports in Japanese men. We report four Japanese male patients with metastatic breast cancer treated with AIs with or without an LH-RH agonist, and consider the relationship between treatment effect and estradiol (E2) concentration. Three patients were initially treated with AI alone after selective estrogen receptor modulators (SERMs), and one received AIs plus an LH-RH agonist after a SERM. Two patients treated with an AI alone responded, one patient with E2 levels below the lower assay limit and the other with levels above the limit. The other treated with an AI alone experienced progression regardless of the E2 levels below the lower assay limit, however, responded after the addition of an LH-RH agonist. E2 concentrations were related to the efficacy of treatment in one patient. The patient initially treated with an AI plus an LH-RH agonist also responded. No grade 3 or 4 adverse events were observed in any of the patients treated with AIs with or without an LH-RH agonist. AIs with or without an LH-RH agonist offer an effective treatment option for hormone receptor-positive metastatic male breast cancer.

  2. Testosterone treatment increases androgen receptor and aromatase gene expression in myotubes from patients with PCOS and controls, but does not induce insulin resistance.

    PubMed

    Eriksen, Mette Brandt; Glintborg, Dorte; Nielsen, Michael Friberg Bruun; Jakobsen, Marianne Antonius; Brusgaard, Klaus; Tan, Qihua; Gaster, Michael

    2014-09-05

    Polycystic ovary syndrome (PCOS) is associated with insulin resistance and increased risk of type 2 diabetes. Skeletal muscle is the major site of insulin mediated glucose disposal and the skeletal muscle tissue is capable to synthesize, convert and degrade androgens. Insulin sensitivity is conserved in cultured myotubes (in vitro) from patients with PCOS, but the effect of testosterone on this insulin sensitivity is unknown. We investigated the effect of 7days testosterone treatment (100nmol/l) on glucose transport and gene expression levels of hormone receptors and enzymes involved in the synthesis and conversion of testosterone (HSD17B1, HSD17B2, CYP19A1, SRD5A1-2, AR, ER-α, HSD17B6 and AKR1-3) in myotubes from ten patients with PCOS and ten matched controls. Testosterone treatment significantly increased aromatase and androgen receptor gene expression levels in patients and controls. Glucose transport in myotubes was comparable in patients with PCOS vs. controls and was unchanged by testosterone treatment (p=0.21 PCOS vs. controls). These results suggest that testosterone treatment of myotubes increases the aromatase and androgen receptor gene expression without affecting insulin sensitivity and if testosterone is implicated in muscular insulin resistance in PCOS, this is by and indirect mechanism.

  3. Natural Product Compounds with Aromatase Inhibitory Activity: An Update

    PubMed Central

    Balunas, Marcy J.; Kinghorn, A. Douglas

    2010-01-01

    Several synthetic aromatase inhibitors are currently in clinical use for the treatment of postmenopausal women with hormone-receptor positive breast cancer. However, these treatments may lead to untoward side effects and so a search for new aromatase inhibitors continues, especially those for which the activity is promoter-specific, targeting the breast-specific promoters I.3 and II. Recently, numerous natural product compounds have been found to inhibit aromatase in non-cellular, cellular, and in vivo studies. These investigations, covering the last two years, as well as additional studies that have focused on the evaluation of natural product compounds as promoter-specific aromatase inhibitors or as aromatase inducers, are described in this review. PMID:20635310

  4. Different Pathways Leading to Integrase Inhibitors Resistance

    PubMed Central

    Thierry, Eloïse; Deprez, Eric; Delelis, Olivier

    2017-01-01

    Integrase strand-transfer inhibitors (INSTIs), such as raltegravir (RAL), elvitegravir, or dolutegravir (DTG), are efficient antiretroviral agents used in HIV treatment in order to inhibit retroviral integration. By contrast to RAL treatments leading to well-identified mutation resistance pathways at the integrase level, recent clinical studies report several cases of patients failing DTG treatment without clearly identified resistance mutation in the integrase gene raising questions for the mechanism behind the resistance. These compounds, by impairing the integration of HIV-1 viral DNA into the host DNA, lead to an accumulation of unintegrated circular viral DNA forms. This viral DNA could be at the origin of the INSTI resistance by two different ways. The first one, sustained by a recent report, involves 2-long terminal repeat circles integration and the second one involves expression of accumulated unintegrated viral DNA leading to a basal production of viral particles maintaining the viral information. PMID:28123383

  5. Inhibition of human CYP19 by azoles used as antifungal agents and aromatase inhibitors, using a new LC-MS/MS method for the analysis of estradiol product formation.

    PubMed

    Trösken, Eva R; Fischer, Kathrin; Völkel, Wolfgang; Lutz, Werner K

    2006-02-15

    Azoles are used as fungicides in agriculture or antifungal drugs in medicine. Their therapeutic activity is based on the inhibition of fungal lanosterol-14alpha-demethylase (CYP51). Azoles are also used for the treatment of estrogen-dependent diseases, e.g. in breast cancer therapy. Inhibition of CYP19 (aromatase) is the working principle for tumor therapy, but is an unwanted side effect of azoles used as fungicides or antifungal drugs. The inhibition of recombinant human CYP19 by 21 azoles in use for the three different purposes was investigated using the natural substrate testosterone. Estradiol product formation was measured by a newly developed and fully validated analytical method based on liquid chromatography-tandem mass spectrometry utilizing photospray ionization (APPI). Potency of enzyme inhibition was expressed in terms of IC50 concentrations. The two cytostatic drugs fadrozole and letrozole were the most potent inhibitors. However, azoles used as fungicides, e.g. prochloraz, or as antifungal drugs, e.g. bifonazole, were almost as potent inhibitors of aromatase as the drugs used in tumor therapy. Comparison of plasma concentrations that may be reached in antifungal therapy do not allow for large safety factors for bifonazole and miconazole. The IC50 values were compared to data obtained with other substrates, such as the pseudo-substrate dibenzylfluorescein (DBF). A high correlation was found, indicating that the fluorescence assay with DBF can well be used for potency ranking and screening of chemicals for aromatase inhibition. The data for antifungal drugs show that side effects on steroid hormone synthesis in humans due to inhibition of aromatase should be considered.

  6. Impact of the estrus cycle and reduction in estrogen levels with aromatase inhibition, on renal function and nitric oxide activity in female rats.

    PubMed

    Santmyire, Beth R; Venkat, Vasuki; Beinder, Ernst; Baylis, Chris

    2010-12-01

    Estradiol increases mRNA and/or protein expression of the nitric oxide synthase (NOS) isoforms in a variety of tissues including kidney. In this study we determined the relationship between cyclical variations in estradiol levels and renal function and total NO production in the virgin female rat. In addition, we used an aromatase inhibitor (Anastrozole), to inhibit synthesis of estradiol from testosterone. Estradiol levels were higher in proestrus vs. diestrus, and were markedly suppressed by 7 days treatment with aromatase inhibitor. There was no difference in total NO production (from urinary and plasma nitrate+nitrite=NO(X)) between proestrus and diestrus but aromatase inhibition resulted in increases in total NO production. The renal cortical NOS activity and protein abundance also increased in aromatase-inhibited female rats. There were no differences in blood pressure (BP) in any group but the renal vascular resistance (RVR) was low in proestrus, increased in diestrus and did not change further after aromatase inhibition. In summary, the cyclical changes in renal function correlate with estradiol but not NO levels. Pharmacologic castration with aromatase inhibition leads to a marked increase in total and renal NOS. This contrasts to earlier work where surgical castration causes decreased NOS.

  7. Reverse the Resistance to PARP Inhibitors

    PubMed Central

    Kim, Yevgeniy; Kim, Aleksei; Sharip, Ainur; Sharip, Aigul; Jiang, Juhong; Yang, Qing; Xie, Yingqiu

    2017-01-01

    One of the DNA repair machineries is activated by Poly (ADP-ribose) Polymerase (PARP) enzyme. Particularly, this enzyme is involved in repair of damages to single-strand DNA, thus decreasing the chances of generating double-strand breaks in the genome. Therefore, the concept to block PARP enzymes by PARP inhibitor (PARPi) was appreciated in cancer treatment. PARPi has been designed and tested for many years and became a potential supplement for the conventional chemotherapy. However, increasing evidence indicates the appearance of the resistance to this treatment. Specifically, cancer cells may acquire new mutations or events that overcome the positive effect of these drugs. This paper describes several molecular mechanisms of PARPi resistance which were reported most recently, and summarizes some strategies to reverse this type of drug resistance. PMID:28255272

  8. Aromatase and leiomyoma of the uterus.

    PubMed

    Shozu, Makio; Murakami, Kouich; Inoue, Masaki

    2004-02-01

    In leiomyoma of the uterus, both aromatase and 17beta-hydroxysteroid dehydrogenase (17beta-HSD) type I are overexpressed compared with myometrium. This suggests that leiomyoma cells convert circulating androstenedione into estrone (via aromatase), then into the active form of estrogen, estradiol (via 17beta-HSD type I). In vitro experiments and several clinical findings support the notion that in situ estrogen plays a role in leiomyoma growth under hypoestrogenemic conditions, such as natural menopause and therapy with gonadotropin-releasing hormone (GnRH) agonists. GnRH agonists abolish estrogen production both in situ in leiomyoma and in the ovary, leading to quick and profound regression of the leiomyoma. Aromatase inhibitors also inhibit estrogen synthesis in both leiomyoma and the ovary and may be used therapeutically. Certain doses of competitive aromatase inhibitors would completely inhibit estrogen production in leiomyoma, whereas ovarian production of estrogen would continue at reduced levels. This may lead to advantageous therapeutic conditions in which leiomyoma regresses without adverse symptoms related to estrogen depletion because levels of ovarian estrogen would be insufficient to support leiomyoma growth but sufficient to prevent symptoms associated with deficiency. This article discusses the potential uses of aromatase inhibitors.

  9. The Effect of COX-2 Inhibitors on the Aromatase Gene (CYP19) Expression in Human Breast Cancer

    DTIC Science & Technology

    2006-12-01

    sulfa ” allergy. Drug Safety 2001; 24 (4), 239-247. 21. Patterson R, Bello A, Lefkowith J: Immunologic tolerability profile of celecoxib... drug is it is associated with less side effects than non- steroidal inflammatory drugs (NSAIDs), particularly gastroduodenal ulcers. Randomized...IIIB disease. 3.8 A woman who has received a COX-2 inhibitor, or NSAID within 7 days of study drug . 3.9 A women who is taking fluconazole, or

  10. Aromatase inhibitor-induced bone loss increases the progression of estrogen receptor-negative breast cancer in bone and exacerbates muscle weakness in vivo.

    PubMed

    Wright, Laura E; Harhash, Ahmed A; Kozlow, Wende M; Waning, David L; Regan, Jenna N; She, Yun; John, Sutha K; Murthy, Sreemala; Niewolna, Maryla; Marks, Andrew R; Mohammad, Khalid S; Guise, Theresa A

    2017-01-31

    Aromatase inhibitors (AIs) cause muscle weakness, bone loss, and joint pain in up to half of cancer patients. Preclinical studies have demonstrated that increased osteoclastic bone resorption can impair muscle contractility and prime the bone microenvironment to accelerate metastatic growth. We hypothesized that AI-induced bone loss could increase breast cancer progression in bone and exacerbate muscle weakness associated with bone metastases. Female athymic nude mice underwent ovariectomy (OVX) or sham surgery and were treated with vehicle or AI (letrozole; Let). An OVX-Let group was then further treated with bisphosphonate (zoledronic acid; Zol). At week three, trabecular bone volume was measured and mice were inoculated with MDA-MB-231 cells into the cardiac ventricle and followed for progression of bone metastases. Five weeks after tumor cell inoculation, tumor-induced osteolytic lesion area was increased in OVX-Let mice and reduced in OVX-Let-Zol mice compared to sham-vehicle. Tumor burden in bone was increased in OVX-Let mice relative to sham-vehicle and OVX-Let-Zol mice. At the termination of the study, muscle-specific force of the extensor digitorum longus muscle was reduced in OVX-Let mice compared to sham-vehicle mice, however, the addition of Zol improved muscle function. In summary, AI treatment induced bone loss and skeletal muscle weakness, recapitulating effects observed in cancer patients. Prevention of AI-induced osteoclastic bone resorption using a bisphosphonate attenuated the development of breast cancer bone metastases and improved muscle function in mice. These findings highlight the bone microenvironment as a modulator of tumor growth locally and muscle function systemically.

  11. Effects of 17α-Methyltestosterone and Aromatase Inhibitor Letrozole on Sex Reversal, Gonadal Structure, and Growth in Yellow Catfish Pelteobagrus fulvidraco.

    PubMed

    Shen, Zhi-Gang; Fan, Qi-Xue; Yang, Wei; Zhang, Yun-Long; Wang, Han-Ping

    2015-04-01

    Monosex populations are in demand in many fish species with sexual dimorphism, e.g., better growth performance, higher gonad value, superior ornamental value. From the point of view of research, a monosex population is one of the best materials for investigating sex-determining mechanisms, sex differentiation, and sex-linked markers. Sex reversal of females (phenotypic reversal from XX female to XX male) is the first step in all-female production in species with an XX/XY system for sex determination. In the present study, masculinization of yellow catfish, a species with XX/XY sex determination, was investigated by oral administration of various doses of 17α-methyltestosterone (MT) or an aromatase inhibitor (AI) letrozole (LZ); effects on survival, growth performance, sex ratio, and changes in gonadal structure were evaluated. Three doses (20, 50, and 100 mg kg(-1) diet) of oral MT or LZ were administered to fry from 10 days post-hatching (DPH) to 59 DPH. Oral administration of MT at all doses did not significantly change the ratio of males (45.8%, 33.3%, and 50.0% respectively) compared to the control group (37.5%), while yielding intersex fish at all doses (4.2% to 8.3%). Oral administration of LZ produced a significantly higher proportion of males in all doses (75.5%, 83.3%, and 75.0%, respectively). Additionally, the lowest dose of LZ improved the growth of treated fish compared to the control, and all doses of LZ enhanced spermatogenesis in treated males.

  12. Evolution of resistance to quorum sensing inhibitors

    PubMed Central

    Kalia, Vipin C.; Wood, Thomas K.; Kumar, Prasun

    2013-01-01

    The major cause of mortality and morbidity in human beings is bacterial infection. Bacteria have developed resistance to most of the antibiotics primarily due to large scale and “indiscriminate” usage. The need is to develop novel mechanisms to treat bacterial infections. The expression of pathogenicity during bacterial infections is mediated by a cell density dependent phenomenon known as quorum sensing (QS). A wide array of QS systems (QSS) is operative in expressing the virulent behavior of bacterial pathogens. Each QSS may be mediated largely by a few major signals along with others produced in minuscule quantities. Efforts to target signal molecules and their receptors have proved effective in alleviating the virulent behavior of such pathogenic bacteria. These QS inhibitors (QSIs) have been reported to be effective in influencing the pathogenicity without affecting bacterial growth. However, evidence is accumulating that bacteria may develop resistance to QSIs. The big question is whether QSIs will meet the same fate as antibiotics? PMID:24194099

  13. Flavonoid inhibition of aromatase enzyme activity in human preadipocytes.

    PubMed

    Campbell, D R; Kurzer, M S

    1993-09-01

    Eleven flavonoid compounds were compared with aminoglutethimide (AG), a pharmaceutical aromatase inhibitor, for their abilities to inhibit aromatase enzyme activity in a human preadipocyte cell culture system. Flavonoids exerting no effect on aromatase activity were catechin, daidzein, equol, genistein, beta-naphthoflavone (BNF), quercetin and rutin. The synthetic flavonoid, alpha-naphthoflavone (ANF), was the most potent aromatase inhibitor, with an I50 value of 0.5 microM. Three naturally-occurring flavonoids, chrysin, flavone, and genistein 4'-methyl ether (Biochanin A) showed I50 values of 4.6, 68, and 113 microM, respectively, while AG showed an I50 value of 7.4 microM. Kinetic analyses showed that both AG and the flavonoids acted as competitive inhibitors of aromatase. The Ki values, indicating the effectiveness of inhibition, were 0.2, 2.4, 2.4, 22, and 49 microM, for ANF, AG, chrysin, flavone, and Biochanin A, respectively. Chrysin, the most potent of the naturally-occurring flavonoids, was similar in potency and effectiveness to AG, a pharmaceutical aromatase inhibitor used clinically in cases of estrogen-dependent carcinoma. These data suggest that flavonoid inhibition of peripheral aromatase activity may contribute to the observed cancer-preventive hormonal effects of plant-based diets.

  14. Inhibitors of aminoglycoside resistance activated in cells.

    PubMed

    Vong, Kenward; Tam, Ingrid S; Yan, Xuxu; Auclair, Karine

    2012-03-16

    The most common mechanism of resistance to aminoglycoside antibiotics entails bacterial expression of drug-metabolizing enzymes, such as the clinically widespread aminoglycoside N-6'-acetyltransferase (AAC(6')). Aminoglycoside-CoA bisubstrates are highly potent AAC(6') inhibitors; however, their inability to penetrate cells precludes in vivo studies. Some truncated bisubstrates are known to cross cell membranes, yet their activities against AAC(6') are in the micromolar range at best. We report here the synthesis and biological activity of aminoglycoside-pantetheine derivatives that, although devoid of AAC(6') inhibitory activity, can potentiate the antibacterial activity of kanamycin A against an aminoglycoside-resistant strain of Enterococcus faecium. Biological studies demonstrate that these molecules are potentially extended to their corresponding full-length bisubstrates by enzymes of the coenzyme A biosynthetic pathway. This work provides a proof-of-concept for the utility of prodrug compounds activated by enzymes of the coenzyme A biosynthetic pathway, to resensitize resistant strains of bacteria to aminoglycoside antibiotics.

  15. Obesity and Postmenopausal Breast Cancer Risk: Determining the Role of Growth Factor-Induced Aromatase Expression

    DTIC Science & Technology

    2013-01-01

    aromatase inhibitor treatment. Obesity is accompanied by elevated levels of growth factors and inflammatory cytokines that can promote tumorigenesis and...whether the greater 8 ASC aromatase expression induced by exposure to Ob MCF-7 CM versus Con (Figure 7A) then results in greater estradiol ...ERE luciferase, when subjects that were on aromatase inhibitor treatment at the time of sera collection were eliminated from the Ob and Con sera

  16. Discovery of a new class of cinnamyl-triazole as potent and selective inhibitors of aromatase (cytochrome P450 19A1).

    PubMed

    McNulty, James; Keskar, Kunal; Crankshaw, Denis J; Holloway, Alison C

    2014-09-15

    Synthesis of a novel class of natural product inspired cinnamyl-containing 1,4,5-triazole and the potent inhibition of human aromatase (CYP 450 19A1) by select members is described. Structure-activity data generated provides insights into the requirements for potency particularly the inclusion of an aryl bromide or chloride residue as a keto-bioisostere.

  17. A phase II study of the histone deacetylase inhibitor vorinostat combined with tamoxifen for the treatment of patients with hormone therapy-resistant breast cancer

    PubMed Central

    Munster, P N; Thurn, K T; Thomas, S; Raha, P; Lacevic, M; Miller, A; Melisko, M; Ismail-Khan, R; Rugo, H; Moasser, M; Minton, S E

    2011-01-01

    Background: Histone deacetylases (HDACs) are crucial components of the oestrogen receptor (ER) transcriptional complex. Preclinically, HDAC inhibitors can reverse tamoxifen/aromatase inhibitor resistance in hormone receptor-positive breast cancer. This concept was examined in a phase II combination trial with correlative end points. Methods: Patients with ER-positive metastatic breast cancer progressing on endocrine therapy were treated with 400 mg of vorinostat daily for 3 of 4 weeks and 20 mg tamoxifen daily, continuously. Histone acetylation and HDAC2 expression in peripheral blood mononuclear cells were also evaluated. Results: In all, 43 patients (median age 56 years (31–71)) were treated, 25 (58%) received prior adjuvant tamoxifen, 29 (67%) failed one prior chemotherapy regimen, 42 (98%) progressed after one, and 23 (54%) after two aromatase inhibitors. The objective response rate by Response Evaluation Criteria in Solid Tumours criteria was 19% and the clinical benefit rate (response or stable disease >24 weeks) was 40%. The median response duration was 10.3 months (confidence interval: 8.1–12.4). Histone hyperacetylation and higher baseline HDAC2 levels correlated with response. Conclusion: The combination of vorinostat and tamoxifen is well tolerated and exhibits encouraging activity in reversing hormone resistance. Correlative studies suggest that HDAC2 expression is a predictive marker and histone hyperacetylation is a useful pharmacodynamic marker for the efficacy of this combination. PMID:21559012

  18. Detecting and treating breast cancer resistance to EGFR inhibitors

    DOEpatents

    Moonlee, Sun-Young; Bissell, Mina J.; Furuta, Saori; Meier, Roland; Kenny, Paraic A.

    2016-04-05

    The application describes therapeutic compositions and methods for treating cancer. For example, therapeutic compositions and methods related to inhibition of FAM83A (family with sequence similarity 83) are provided. The application also describes methods for diagnosing cancer resistance to EGFR inhibitors. For example, a method of diagnosing cancer resistance to EGFR inhibitors by detecting increased FAM83A levels is described.

  19. Aromatase regulates aggression in the African cichlid fish Astatotilapia burtoni.

    PubMed

    Huffman, Lin S; O'Connell, Lauren A; Hofmann, Hans A

    2013-03-15

    The roles of estrogen and androgens in male social behavior are well studied, but little is known about how these hormones contribute to behavior in a social hierarchy. Here we test the role of aromatase, the enzyme that converts testosterone into estradiol, in mediating aggression and reproductive behavior in male Astatotilapia burtoni, an African cichlid fish that displays remarkable plasticity in social behavior. We first measured aromatase expression in subordinate and dominant males in brain regions that regulate social behavior and found that subordinate males have higher aromatase expression than dominant males in the magnocellular and gigantocellular regions of the preoptic area. Next, we functionally tested the role of aromatase in regulating behavior by intraperitoneally injecting dominant males with either saline or fadrozole (FAD), an aromatase inhibitor, and found that FAD treatment decreases aggressive, but not reproductive, behaviors compared to saline controls. To determine the underlying physiological and molecular consequences of FAD treatment, we measured estradiol and testosterone levels from plasma and brain aromatase expression in FAD and saline treated dominant males. We found that estradiol levels decreased and testosterone levels increased in response to FAD treatment. Moreover, FAD treated males had increased aromatase expression in the gigantocellular portion of the POA, possibly a compensatory response. Overall, our results suggest aromatase is a key enzyme that promotes aggression in A. burtoni males through actions in the preoptic area.

  20. Combining computational and biochemical studies for a rationale on the anti-aromatase activity of natural polyphenols.

    PubMed

    Neves, Marco A C; Dinis, Teresa C P; Colombo, Giorgio; Sá e Melo, M Luisa

    2007-12-01

    Aromatase, an enzyme of the cytochrome P450 family, is a very important pharmacological target, particularly for the treatment of breast cancer. The anti-aromatase activity of a set of natural polyphenolic compounds was evaluated in vitro. Strong aromatase inhibitors including flavones, flavanones, resveratrol, and oleuropein, with activities comparable to that of the reference anti-aromatase drug aminoglutethimide, were identified. Through the application of molecular modeling techniques based on grid-independent descriptors and molecular interaction fields, the major physicochemical features associated with inhibitory activity were disclosed, and a putative virtual active site of aromatase was proposed. Docking of the inhibitors into a 3D homology model structure of the enzyme defined a common binding mode for the small molecules under investigation. The good correlation between computational and biological results provides the first rationalization of the anti-aromatase activity of polyphenolic compounds. Moreover, the information generated in this approach should be further exploited for the design of new aromatase inhibitors.

  1. Ki67 Proliferation Index as a Tool for Chemotherapy Decisions During and After Neoadjuvant Aromatase Inhibitor Treatment of Breast Cancer: Results From the American College of Surgeons Oncology Group Z1031 Trial (Alliance).

    PubMed

    Ellis, Matthew J; Suman, Vera J; Hoog, Jeremy; Goncalves, Rodrigo; Sanati, Souzan; Creighton, Chad J; DeSchryver, Katherine; Crouch, Erika; Brink, Amy; Watson, Mark; Luo, Jingqin; Tao, Yu; Barnes, Michael; Dowsett, Mitchell; Budd, G Thomas; Winer, Eric; Silverman, Paula; Esserman, Laura; Carey, Lisa; Ma, Cynthia X; Unzeitig, Gary; Pluard, Timothy; Whitworth, Pat; Babiera, Gildy; Guenther, J Michael; Dayao, Zoneddy; Ota, David; Leitch, Marilyn; Olson, John A; Allred, D Craig; Hunt, Kelly

    2017-04-01

    Purpose To determine the pathologic complete response (pCR) rate in estrogen receptor (ER) -positive primary breast cancer triaged to chemotherapy when the protein encoded by the MKI67 gene (Ki67) level was > 10% after 2 to 4 weeks of neoadjuvant aromatase inhibitor (AI) therapy. A second objective was to examine risk of relapse using the Ki67-based Preoperative Endocrine Prognostic Index (PEPI). Methods The American College of Surgeons Oncology Group (ACOSOG) Z1031A trial enrolled postmenopausal women with stage II or III ER-positive (Allred score, 6 to 8) breast cancer whose treatment was randomly assigned to neoadjuvant AI therapy with anastrozole, exemestane, or letrozole. For the trial ACOSOG Z1031B, the protocol was amended to include a tumor Ki67 determination after 2 to 4 weeks of AI. If the Ki67 was > 10%, patients were switched to neoadjuvant chemotherapy. A pCR rate of > 20% was the predefined efficacy threshold. In patients who completed neoadjuvant AI, stratified Cox modeling was used to assess whether time to recurrence differed by PEPI = 0 score (T1 or T2, N0, Ki67 < 2.7%, ER Allred > 2) versus PEPI > 0 disease. Results Only two of the 35 patients in ACOSOG Z1031B who were switched to neoadjuvant chemotherapy experienced a pCR (5.7%; 95% CI, 0.7% to 19.1%). After 5.5 years of median follow-up, four (3.7%) of the 109 patients with a PEPI = 0 score relapsed versus 49 (14.4%) of 341 of patients with PEPI > 0 (recurrence hazard ratio [PEPI = 0 v PEPI > 0], 0.27; P = .014; 95% CI, 0.092 to 0.764). Conclusion Chemotherapy efficacy was lower than expected in ER-positive tumors exhibiting AI-resistant proliferation. The optimal therapy for these patients should be further investigated. For patients with PEPI = 0 disease, the relapse risk over 5 years was only 3.6% without chemotherapy, supporting the study of adjuvant endocrine monotherapy in this group. These Ki67 and PEPI triage approaches are being definitively studied in the ALTERNATE trial (Alternate

  2. Aromatase excess in cancers of breast, endometrium and ovary.

    PubMed

    Bulun, Serdar E; Chen, Dong; Lu, Meiling; Zhao, Hong; Cheng, Youhong; Demura, Masashi; Yilmaz, Bertan; Martin, Regina; Utsunomiya, Hiroki; Thung, Steven; Su, Emily; Marsh, Erica; Hakim, Amy; Yin, Ping; Ishikawa, Hiroshi; Amin, Sanober; Imir, Gonca; Gurates, Bilgin; Attar, Erkut; Reierstad, Scott; Innes, Joy; Lin, Zhihong

    2007-01-01

    Pathogenesis and growth of three common women's cancers (breast, endometrium and ovary) are linked to estrogen. A single gene encodes the key enzyme for estrogen biosynthesis named aromatase, inhibition of which effectively eliminates estrogen production in the entire body. Aromatase inhibitors successfully treat breast cancer, whereas their roles in endometrial and ovarian cancers are less clear. Ovary, testis, adipose tissue, skin, hypothalamus and placenta express aromatase normally, whereas breast, endometrial and ovarian cancers overexpress aromatase and produce local estrogen exerting paracrine and intracrine effects. Tissue-specific promoters distributed over a 93-kb regulatory region upstream of a common coding region alternatively control aromatase expression. A distinct set of transcription factors regulates each promoter in a signaling pathway- and tissue-specific manner. In cancers of breast, endometrium and ovary, aromatase expression is primarly regulated by increased activity of the proximally located promoter I.3/II region. Promoters I.3 and II lie 215 bp from each other and are coordinately stimulated by PGE(2) via a cAMP-PKA-dependent pathway. In breast adipose fibroblasts exposed to PGE(2) secreted by malignant epithelial cells, PKC is also activated, and this potentiates cAMP-PKA-dependent induction of aromatase. Thus, inflammatory substances such as PGE(2) may play important roles in inducing local production of estrogen that promotes tumor growth.

  3. Resistance to anti-influenza drugs: adamantanes and neuraminidase inhibitors.

    PubMed

    Hurt, Aeron C; Ho, Hui-Ting; Barr, Ian

    2006-10-01

    Development of effective drugs for the treatment or prevention of epidemic and pandemic influenza is important in order to reduce its impact. Adamantanes and neuraminidase inhibitors are two classes of anti-influenza drugs available for influenza therapy currently. However, emergence of resistance to these drugs has been detected, which raises concerns regarding their widespread use. In this review, resistance to the adamantanes and neuraminidase inhibitors will be discussed in relation to both epidemic and pandemic influenza viruses.

  4. Potential utility of natural products as regulators of breast cancer-associated aromatase promoters

    PubMed Central

    2011-01-01

    Aromatase, the key enzyme in estrogen biosynthesis, converts androstenedione to estrone and testosterone to estradiol. The enzyme is expressed in various tissues such as ovary, placenta, bone, brain, skin, and adipose tissue. Aromatase enzyme is encoded by a single gene CYP 19A1 and its expression is controlled by tissue-specific promoters. Aromatase mRNA is primarily transcribed from promoter I.4 in normal breast tissue and physiological levels of aromatase are found in breast adipose stromal fibroblasts. Under the conditions of breast cancer, as a result of the activation of a distinct set of aromatase promoters (I.3, II, and I.7) aromatase expression is enhanced leading to local overproduction of estrogen that promotes breast cancer. Aromatase is considered as a potential target for endocrine treatment of breast cancer but due to nonspecific reduction of aromatase activity in other tissues, aromatase inhibitors (AIs) are associated with undesirable side effects such as bone loss, and abnormal lipid metabolism. Inhibition of aromatase expression by inactivating breast tumor-specific aromatase promoters can selectively block estrogen production at the tumor site. Although several synthetic chemical compounds and nuclear receptor ligands are known to inhibit the activity of the tumor-specific aromatase promoters, further development of more specific and efficacious drugs without adverse effects is still warranted. Plants are rich in chemopreventive agents that have a great potential to be used in chemotherapy for hormone dependent breast cancer which could serve as a source for natural AIs. In this brief review, we summarize the studies on phytochemicals such as biochanin A, genistein, quercetin, isoliquiritigenin, resveratrol, and grape seed extracts related to their effect on the activation of breast cancer-associated aromatase promoters and discuss their aromatase inhibitory potential to be used as safer chemotherapeutic agents for specific hormone

  5. Potential utility of natural products as regulators of breast cancer-associated aromatase promoters.

    PubMed

    Khan, Shabana I; Zhao, Jianping; Khan, Ikhlas A; Walker, Larry A; Dasmahapatra, Asok K

    2011-06-21

    Aromatase, the key enzyme in estrogen biosynthesis, converts androstenedione to estrone and testosterone to estradiol. The enzyme is expressed in various tissues such as ovary, placenta, bone, brain, skin, and adipose tissue. Aromatase enzyme is encoded by a single gene CYP 19A1 and its expression is controlled by tissue-specific promoters. Aromatase mRNA is primarily transcribed from promoter I.4 in normal breast tissue and physiological levels of aromatase are found in breast adipose stromal fibroblasts. Under the conditions of breast cancer, as a result of the activation of a distinct set of aromatase promoters (I.3, II, and I.7) aromatase expression is enhanced leading to local overproduction of estrogen that promotes breast cancer. Aromatase is considered as a potential target for endocrine treatment of breast cancer but due to nonspecific reduction of aromatase activity in other tissues, aromatase inhibitors (AIs) are associated with undesirable side effects such as bone loss, and abnormal lipid metabolism. Inhibition of aromatase expression by inactivating breast tumor-specific aromatase promoters can selectively block estrogen production at the tumor site. Although several synthetic chemical compounds and nuclear receptor ligands are known to inhibit the activity of the tumor-specific aromatase promoters, further development of more specific and efficacious drugs without adverse effects is still warranted. Plants are rich in chemopreventive agents that have a great potential to be used in chemotherapy for hormone dependent breast cancer which could serve as a source for natural AIs. In this brief review, we summarize the studies on phytochemicals such as biochanin A, genistein, quercetin, isoliquiritigenin, resveratrol, and grape seed extracts related to their effect on the activation of breast cancer-associated aromatase promoters and discuss their aromatase inhibitory potential to be used as safer chemotherapeutic agents for specific hormone

  6. Canine osteosarcoma cells exhibit resistance to aurora kinase inhibitors.

    PubMed

    Cannon, C M; Pozniak, J; Scott, M C; Ito, D; Gorden, B H; Graef, A J; Modiano, J F

    2015-03-01

    We evaluated the effect of Aurora kinase inhibitors AZD1152 and VX680 on canine osteosarcoma cells. Cytotoxicity was seen in all four cell lines; however, half-maximal inhibitory concentrations were significantly higher than in human leukaemia and canine lymphoma cells. AZD1152 reduced Aurora kinase B phosphorylation, indicating resistance was not because of failure of target recognition. Efflux mediated by ABCB1 and ABCG2 transporters is one known mechanism of resistance against these drugs and verapamil enhanced AZD1152-induced apoptosis; however, these transporters were only expressed by a small percentage of cells in each line and the effects of verapamil were modest, suggesting other mechanisms contribute to resistance. Our results indicate that canine osteosarcoma cells are resistant to Aurora kinase inhibitors and suggest that these compounds are unlikely to be useful as single agents for this disease. Further investigation of these resistance mechanisms and the potential utility of Aurora kinase inhibitors in multi-agent protocols is warranted.

  7. Development of PPO inhibitor-resistant cultures and crops.

    PubMed

    Li, Xianggan; Nicholl, David

    2005-03-01

    Recent progress in the development of protoporphyrinogen oxidase (PPO, Protox) inhibitor-resistant plant cell cultures and crops is reviewed, with emphasis on the molecular and cellular aspects of this topic. PPO herbicide-resistant maize plants have been reported, along with the isolation of plant PPO genes and the isolation of herbicide-resistant mutants. At the same time, PPO inhibitor-resistant rice plants have been developed by expression of the Bacillus subtilis PPO gene via targeting the gene into either chloroplast or cytoplasm. Other attempts to develop PPO herbicide-resistant plants include conventional tissue culture methods, expression of modified co-factors of the protoporphyrin IX binding subunit proteins, over-expression of wild-type plant PPO gene, and engineering of P-450 monooxygenases to degrade the PPO inhibitor.

  8. Resistance to BRAF inhibitors induces glutamine dependency in melanoma cells

    PubMed Central

    Baenke, Franziska; Chaneton, Barbara; Smith, Matthew; Van Den Broek, Niels; Hogan, Kate; Tang, Haoran; Viros, Amaya; Martin, Matthew; Galbraith, Laura; Girotti, Maria R.; Dhomen, Nathalie; Gottlieb, Eyal; Marais, Richard

    2016-01-01

    BRAF inhibitors can extend progression-free and overall survival in melanoma patients whose tumors harbor mutations in BRAF. However, the majority of patients eventually develop resistance to these drugs. Here we show that BRAF mutant melanoma cells that have developed acquired resistance to BRAF inhibitors display increased oxidative metabolism and increased dependency on mitochondria for survival. Intriguingly, the increased oxidative metabolism is associated with a switch from glucose to glutamine metabolism and an increased dependence on glutamine over glucose for proliferation. We show that the resistant cells are more sensitive to mitochondrial poisons and to inhibitors of glutaminolysis, suggesting that targeting specific metabolic pathways may offer exciting therapeutic opportunities to treat resistant tumors, or to delay emergence of resistance in the first-line setting. PMID:26365896

  9. Synthesis and aromatase inhibitory activity of novel pyridine-containing isoflavones.

    PubMed

    Kim, Young-Woo; Hackett, John C; Brueggemeier, Robert W

    2004-07-29

    Aromatase, a cytochrome P450 hemoprotein that is responsible for estrogen biosynthesis by conversion of androgens into estrogens, has been an attractive target in the treatment of hormone-dependent breast cancer. As a result, a number of synthetic steroidal or nonsteroidal aromatase inhibitors have been successfully developed. In addition, there are several classes of natural products that exert potent activities in aromatase inhibition, with the flavonoids being most prominent. Previous studies have exploited flavone and flavanone scaffolds for the development of new aromatase inhibitors. In this paper, we describe the design, synthesis, and biological evaluation of a novel series of 2-(4'-pyridylmethyl)thioisoflavones as the first example of synthetic isoflavone-based aromatase inhibitors.

  10. Modulation of aromatase activity as a mode of action for endocrine disrupting chemicals in a marine fish.

    PubMed

    Mills, Lesley J; Gutjahr-Gobell, Ruth E; Zaroogian, Gerald E; Horowitz, Doranne Borsay; Laws, Susan C

    2014-02-01

    The steroidogenic enzyme aromatase catalyzes the conversion of androgens to estrogens and therefore plays a central role in reproduction. In contrast to most vertebrates, teleost fish have two distinct forms of aromatase. Because brain aromatase activity in fish is up to 1000 times that in mammals, fish may be especially susceptible to negative effects from environmental endocrine-disrupting chemicals (EDCs) that impact aromatase activity. In this study, the effects of estradiol (E2), ethynylestradiol (EE2), octylphenol (OP), and androstatrienedione (ATD) on reproduction and aromatase activity in brains and gonads from the marine fish cunner (Tautogolabrus adspersus) was investigated. The purpose of the study was to explore the relationship between changes in aromatase activity and reproductive output in a marine fish, as well as compare aromatase activity to two commonly used indicators of EDC exposure, plasma vitellogenin (VTG) and gonadosomatic index (GSI). Results with E2, EE2, and ATD indicate that aromatase activity in cunner brain and ovary are affected differently by exposure to these EDCs. In the case of E2 and EE2, male brain aromatase activity was signficantly increased by these treatments, female brain aromatase activity was unaffected, and ovarian aromatase activity was significantly decreased. Treatment with the aromatase inhibitor ATD resulted in significantly decreased aromatase activity in male and female brain, but had no significant impact on ovarian aromatase activity. Regardless of test chemical, a decrease or an increase in male brain aromatase activity relative to controls was associated with decreased egg production in cunner and was also correlated with significant changes in GSI in both sexes. E2 and EE2 significantly elevated plasma VTG in males and females, while ATD had no significant effect. Treatment of cunner with OP had no significant effect on any measured endpoint. Overall, results with these exposures indicate EDCs that impact

  11. Design and Synthesis of Norendoxifen Analogues with Dual Aromatase Inhibitory and Estrogen Receptor Modulatory Activities

    PubMed Central

    Lv, Wei; Liu, Jinzhong; Skaar, Todd C.; Flockhart, David A.; Cushman, Mark

    2015-01-01

    Both selective estrogen receptor modulators and aromatase inhibitors are widely used for the treatment of breast cancer. Compounds with both aromatase inhibitory and estrogen receptor modulatory activities could have special advantages for treatment of breast cancer. Our previous efforts led to the discovery of norendoxifen as the first compound with dual aromatase inhibitory and estrogen receptor binding activities. To optimize its efficacy and aromatase selectivity versus other cytochrome P450 enzymes, a series of structurally related norendoxifen analogues were designed and synthesized. The most potent compound, 4'-hydroxynorendoxifen (10), displayed elevated inhibitory potency against aromatase and enhanced affinity for estrogen receptors when compared to norendoxifen. The selectivity of 10 for aromatase versus other cytochrome P450 enzymes was also superior to norendoxifen. 4'-Hydroxynorendoxifen is therefore an interesting lead for further development to obtain new anticancer agents of potential value for the treatment of breast cancer. PMID:25751283

  12. Impact of Aromatase protein variants and drug interactions in breast cancer: a molecular docking approach.

    PubMed

    Setti, Aravind; Venugopal Rao, V; Priyamvada Devi, A; Pawar, Smita C; Naresh, B; Kalyan, C S V V

    2012-08-01

    Breast cancer is a frequently reported cancer in women all over the world. Several methods available to cure the breast cancer based on stage. This study focused on chemoprevention drugs of Aromatase, a potential target in breast cancer. Natural variants of Aromatase are very common; they have been collected and modeled, optimized the energy of mutated Aromatase protein. Reversible (Anastrozole) and irreversible (Exemestane) Aromatase inhibitors are selected and performed molecular docking studies of each drug against each variant to see the binding affinity impact on protein variant and drugs. In this comparative study, Anastrozole, a cumene derivative showed more binding affinity and Diethylstilbestrol showed weak binding affinity against among all drugs. The comparative molecular docking revealed that the binding affinity between drug and Aromatase protein variant is imprecise but fairly close; therefore the protein variants of Aromatase can be conceived to be equal for chemoprevention of breast cancer therapy.

  13. White button mushroom phytochemicals inhibit aromatase activity and breast cancer cell proliferation.

    PubMed

    Grube, B J; Eng, E T; Kao, Y C; Kwon, A; Chen, S

    2001-12-01

    Estrogen is a major factor in the development of breast cancer. In situ estrogen production by aromatase/estrogen synthetase in breast cancer plays a dominant role in tumor proliferation. Because natural compounds such as flavones and isoflavones have been shown to be inhibitors of aromatase, it is thought that vegetables that contain these phytochemicals can inhibit aromatase activity and suppress breast cancer cell proliferation. Heat-stable extracts were prepared from vegetables and screened for their ability to inhibit aromatase activity in a human placental microsome assay. The white button mushroom (species Agaricus bisporus) suppressed aromatase activity dose dependently. Enzyme kinetics demonstrated mixed inhibition, suggesting the presence of multiple inhibitors or more than one inhibitory mechanism. "In cell" aromatase activity and cell proliferation were measured using MCF-7aro, an aromatase-transfected breast cancer cell line. Phytochemicals in the mushroom aqueous extract inhibited aromatase activity and proliferation of MCF-7aro cells. These results suggest that diets high in mushrooms may modulate the aromatase activity and function in chemoprevention in postmenopausal women by reducing the in situ production of estrogen.

  14. Novel proteinase inhibitor promotes resistance to insects

    Technology Transfer Automated Retrieval System (TEKTRAN)

    A novel Beta vulgaris serine proteinase inhibitor gene (BvSTI) and its protein are identified in response to insect feeding on B. vulgaris seedlings. BvSTI is cloned into an expression vector with constitutive promoter and transformed into Nicotiana benthamiana plants to assess BvSTI’s ability to ...

  15. [Mechanisms of resistance to BCR-ABL kinase inhibitors].

    PubMed

    Diamond, Joana; da Silva, Maria Gomes

    2013-01-01

    Since the introduction of imatinib mesylate for the treatment of chronic myeloid leukaemia, impressive clinical responses were observed in the majority of patients in chronic phase. However, not all patients experience an optimal response to imatinib mesylate or even to the more potent, second generation tyrosine kinase inhibitors. Furthermore, responses are not sustained in a number of patients, and it is yet unclear whether the inhibitors can be safely discontinued in patients who achieve long-term remission. The emergence of resistance to second generation tyrosine kinase inhibitors has become a significant problem that led to extensive studies on the causal mechanisms. This review will describe our current state of knowledge on why and how chronic myeloid leukaemia cells can develop resistance to second generation tyrosine kinase inhibitors.

  16. Exploring new chemical functionalities to improve aromatase inhibition of steroids.

    PubMed

    Varela, Carla L; Amaral, Cristina; Correia-da-Silva, Georgina; Costa, Saul C; Carvalho, Rui A; Costa, Giosuè; Alcaro, Stefano; Teixeira, Natércia A A; Tavares-da-Silva, Elisiário J; Roleira, Fernanda M F

    2016-06-15

    In this work, new potent steroidal aromatase inhibitors both in microsomes and in breast cancer cells have been found. The synthesis of the 3,4-(ethylenedioxy)androsta-3,5-dien-17-one (12), a new steroid containing a heterocycle dioxene fused in the A-ring, led to the discovery of a new reaction for which a mechanism is proposed. New structure-activity relationships were established. Some 5β-steroids, such as compound 4β,5β-epoxyandrostan-17-one (9), showed aromatase inhibitory activity, because they adopt a similar A-ring conformation as those of androstenedione, the natural substrate of aromatase. Moreover, new chemical features to increase planarity were disclosed, specifically the 3α,4α-cyclopropane ring, as in 3α,4α-methylen-5α-androstan-17-one (5) (IC50=0.11μM), and the Δ(9-11) double bond in the C-ring, as in androsta-4,9(11)-diene-3,17-dione (13) (IC50=0.25μM). In addition, induced-fit docking (IFD) simulations and site of metabolism (SoM) predictions helped to explain the recognition of new potent steroidal aromatase inhibitors within the enzyme. These insights can be valuable tools for the understanding of the molecular recognition process by the aromatase and for the future design of new steroidal inhibitors.

  17. BET inhibitor resistance emerges from leukaemia stem cells.

    PubMed

    Fong, Chun Yew; Gilan, Omer; Lam, Enid Y N; Rubin, Alan F; Ftouni, Sarah; Tyler, Dean; Stanley, Kym; Sinha, Devbarna; Yeh, Paul; Morison, Jessica; Giotopoulos, George; Lugo, Dave; Jeffrey, Philip; Lee, Stanley Chun-Wei; Carpenter, Christopher; Gregory, Richard; Ramsay, Robert G; Lane, Steven W; Abdel-Wahab, Omar; Kouzarides, Tony; Johnstone, Ricky W; Dawson, Sarah-Jane; Huntly, Brian J P; Prinjha, Rab K; Papenfuss, Anthony T; Dawson, Mark A

    2015-09-24

    Bromodomain and extra terminal protein (BET) inhibitors are first-in-class targeted therapies that deliver a new therapeutic opportunity by directly targeting bromodomain proteins that bind acetylated chromatin marks. Early clinical trials have shown promise, especially in acute myeloid leukaemia, and therefore the evaluation of resistance mechanisms is crucial to optimize the clinical efficacy of these drugs. Here we use primary mouse haematopoietic stem and progenitor cells immortalized with the fusion protein MLL-AF9 to generate several single-cell clones that demonstrate resistance, in vitro and in vivo, to the prototypical BET inhibitor, I-BET. Resistance to I-BET confers cross-resistance to chemically distinct BET inhibitors such as JQ1, as well as resistance to genetic knockdown of BET proteins. Resistance is not mediated through increased drug efflux or metabolism, but is shown to emerge from leukaemia stem cells both ex vivo and in vivo. Chromatin-bound BRD4 is globally reduced in resistant cells, whereas the expression of key target genes such as Myc remains unaltered, highlighting the existence of alternative mechanisms to regulate transcription. We demonstrate that resistance to BET inhibitors, in human and mouse leukaemia cells, is in part a consequence of increased Wnt/β-catenin signalling, and negative regulation of this pathway results in restoration of sensitivity to I-BET in vitro and in vivo. Together, these findings provide new insights into the biology of acute myeloid leukaemia, highlight potential therapeutic limitations of BET inhibitors, and identify strategies that may enhance the clinical utility of these unique targeted therapies.

  18. PP2A Inhibitor PME-1 Drives Kinase Inhibitor Resistance in Glioma Cells.

    PubMed

    Kaur, Amanpreet; Denisova, Oxana V; Qiao, Xi; Jumppanen, Mikael; Peuhu, Emilia; Ahmed, Shafiq U; Raheem, Olayinka; Haapasalo, Hannu; Eriksson, John; Chalmers, Anthony J; Laakkonen, Pirjo; Westermarck, Jukka

    2016-12-01

    Glioblastoma multiforme lacks effective therapy options. Although deregulated kinase pathways are drivers of malignant progression in glioblastoma multiforme, glioma cells exhibit intrinsic resistance toward many kinase inhibitors, and the molecular basis of this resistance remains poorly understood. Here, we show that overexpression of the protein phosphatase 2A (PP2A) inhibitor protein PME-1 drives resistance of glioma cells to various multikinase inhibitors. The PME-1-elicited resistance was dependent on specific PP2A complexes and was mediated by a decrease in cytoplasmic HDAC4 activity. Importantly, both PME-1 and HDAC4 associated with human glioma progression, supporting clinical relevance of the identified mechanism. Synthetic lethality induced by both PME-1 and HDAC4 inhibition was dependent on the coexpression of proapoptotic protein BAD. Thus, PME-1-mediated PP2A inhibition is a novel mechanistic explanation for multikinase inhibitor resistance in glioma cells. Clinically, these results may inform patient stratification strategies for future clinical trials with selected kinase inhibitors in glioblastoma multiforme. Cancer Res; 76(23); 7001-11. ©2016 AACR.

  19. Effects of flavonoids on aromatase activity, an in vitro study.

    PubMed

    Pelissero, C; Lenczowski, M J; Chinzi, D; Davail-Cuisset, B; Sumpter, J P; Fostier, A

    1996-02-01

    In the study, the inhibitory effect of flavonoids, including isoflavonic phytoestrogens, on the ovarian aromatase enzyme complex from the rainbow trout, Oncorhynchus mykiss, was assessed in vitro. Some of the compounds tested on fish were also tested on human placental aromatase activity as a comparison between the two sources of enzyme. It was found that flavone, dl-aminoglutethimide, apigenin, quercetin, 7,4'- dihydroxyflavone, alpha-naphthoflavone and equol were potent inhibitors of the ovarian aromatase activity in rainbow trout. Relative potencies (RP) of these compounds compared to flavone (assigned an effect of 1) were, respectively, 19.0, 8.7, 5.3, 3.7, 3.2 and 0.9. Two other phytoestrogens, namely biochanin A and genistein, slightly inhibited aromatase activity. Finally, 7-hydroxyflavone, formononetin, daidzein, coumestrol, chrysin, flavanone and estradiol-17beta did not inhibit ovarian aromatase activity at doses up to 1000 microM. Experiments on human placental aromatase showed inhibitory effects of dl-aminoglutethimide, flavone, flavanone and equol with RP values of 2.8. 1, 1.5 and 0.4, respectively. These results are in accordance with previous studies. The influence of the experimental procedure on IC50 values and RP is discussed.

  20. Development of covalent inhibitors that can overcome resistance to first-generation FGFR kinase inhibitors.

    PubMed

    Tan, Li; Wang, Jun; Tanizaki, Junko; Huang, Zhifeng; Aref, Amir R; Rusan, Maria; Zhu, Su-Jie; Zhang, Yiyun; Ercan, Dalia; Liao, Rachel G; Capelletti, Marzia; Zhou, Wenjun; Hur, Wooyoung; Kim, NamDoo; Sim, Taebo; Gaudet, Suzanne; Barbie, David A; Yeh, Jing-Ruey Joanna; Yun, Cai-Hong; Hammerman, Peter S; Mohammadi, Moosa; Jänne, Pasi A; Gray, Nathanael S

    2014-11-11

    The human FGF receptors (FGFRs) play critical roles in various human cancers, and several FGFR inhibitors are currently under clinical investigation. Resistance usually results from selection for mutant kinases that are impervious to the action of the drug or from up-regulation of compensatory signaling pathways. Preclinical studies have demonstrated that resistance to FGFR inhibitors can be acquired through mutations in the FGFR gatekeeper residue, as clinically observed for FGFR4 in embryonal rhabdomyosarcoma and neuroendocrine breast carcinomas. Here we report on the use of a structure-based drug design to develop two selective, next-generation covalent FGFR inhibitors, the FGFR irreversible inhibitors 2 (FIIN-2) and 3 (FIIN-3). To our knowledge, FIIN-2 and FIIN-3 are the first inhibitors that can potently inhibit the proliferation of cells dependent upon the gatekeeper mutants of FGFR1 or FGFR2, which confer resistance to first-generation clinical FGFR inhibitors such as NVP-BGJ398 and AZD4547. Because of the conformational flexibility of the reactive acrylamide substituent, FIIN-3 has the unprecedented ability to inhibit both the EGF receptor (EGFR) and FGFR covalently by targeting two distinct cysteine residues. We report the cocrystal structure of FGFR4 with FIIN-2, which unexpectedly exhibits a "DFG-out" covalent binding mode. The structural basis for dual FGFR and EGFR targeting by FIIN3 also is illustrated by crystal structures of FIIN-3 bound with FGFR4 V550L and EGFR L858R. These results have important implications for the design of covalent FGFR inhibitors that can overcome clinical resistance and provide the first example, to our knowledge, of a kinase inhibitor that covalently targets cysteines located in different positions within the ATP-binding pocket.

  1. Development of covalent inhibitors that can overcome resistance to first-generation FGFR kinase inhibitors

    PubMed Central

    Tan, Li; Wang, Jun; Tanizaki, Junko; Huang, Zhifeng; Aref, Amir R.; Rusan, Maria; Zhu, Su-Jie; Zhang, Yiyun; Ercan, Dalia; Liao, Rachel G.; Capelletti, Marzia; Zhou, Wenjun; Hur, Wooyoung; Kim, NamDoo; Sim, Taebo; Gaudet, Suzanne; Barbie, David A.; Yeh, Jing-Ruey Joanna; Yun, Cai-Hong; Hammerman, Peter S.; Mohammadi, Moosa; Jänne, Pasi A.; Gray, Nathanael S.

    2014-01-01

    The human FGF receptors (FGFRs) play critical roles in various human cancers, and several FGFR inhibitors are currently under clinical investigation. Resistance usually results from selection for mutant kinases that are impervious to the action of the drug or from up-regulation of compensatory signaling pathways. Preclinical studies have demonstrated that resistance to FGFR inhibitors can be acquired through mutations in the FGFR gatekeeper residue, as clinically observed for FGFR4 in embryonal rhabdomyosarcoma and neuroendocrine breast carcinomas. Here we report on the use of a structure-based drug design to develop two selective, next-generation covalent FGFR inhibitors, the FGFR irreversible inhibitors 2 (FIIN-2) and 3 (FIIN-3). To our knowledge, FIIN-2 and FIIN-3 are the first inhibitors that can potently inhibit the proliferation of cells dependent upon the gatekeeper mutants of FGFR1 or FGFR2, which confer resistance to first-generation clinical FGFR inhibitors such as NVP-BGJ398 and AZD4547. Because of the conformational flexibility of the reactive acrylamide substituent, FIIN-3 has the unprecedented ability to inhibit both the EGF receptor (EGFR) and FGFR covalently by targeting two distinct cysteine residues. We report the cocrystal structure of FGFR4 with FIIN-2, which unexpectedly exhibits a “DFG-out” covalent binding mode. The structural basis for dual FGFR and EGFR targeting by FIIN3 also is illustrated by crystal structures of FIIN-3 bound with FGFR4 V550L and EGFR L858R. These results have important implications for the design of covalent FGFR inhibitors that can overcome clinical resistance and provide the first example, to our knowledge, of a kinase inhibitor that covalently targets cysteines located in different positions within the ATP-binding pocket. PMID:25349422

  2. Randomized controlled trial of toremifene 120 mg compared with exemestane 25 mg after prior treatment with a non-steroidal aromatase inhibitor in postmenopausal women with hormone receptor-positive metastatic breast cancer

    PubMed Central

    2013-01-01

    Background After the failure of a non-steroidal aromatase inhibitor (nsAI) for postmenopausal patients with metastatic breast cancer (mBC), it is unclear which of various kinds of endocrine therapy is the most appropriate. A randomized controlled trial was performed to compare the efficacy and safety of daily toremifene 120 mg (TOR120), a selective estrogen receptor modulator, and exemestane 25 mg (EXE), a steroidal aromatase inhibitor. The primary end point was the clinical benefit rate (CBR). The secondary end points were objective response rate (ORR), progression-free survival (PFS), overall survival (OS) and toxicity. Methods Initially, a total of 91 women was registered in the study and randomly assigned to either TOR120 (n = 46) or EXE (n = 45) from October 2008 to November 2011. Three of the 46 patients in the TOR120 arm were not received treatment, 2 patients having withdrawn from the trial by their preference and one having been dropped due to administration of another SERM. Results When analyzed after a median observation period of 16.9 months, the intention-to-treat analysis showed that there were no statistical difference between TOR120 (N = 46) and EXE (n = 45) in terms of CBR (41.3% vs. 26.7%; P = 0.14), ORR (10.8% vs. 2.2%; P = 0.083), and OS (Hazard ratio, 0.60; P = 0.22). The PFS of TOR120 was longer than that of EXE, the difference being statistically significant (Hazard ratio, 0.61, P = 0.045). The results in treatment-received cohort (N = 88) were similar to those in ITT cohort. Both treatments were well-tolerated with no severe adverse events, although the treatment of 3 of 43 women administered TOR120 was stopped after a few days because of nausea, general fatigue, hot flush and night sweating. Conclusions TOR120, as a subsequent endocrine therapy for mBC patients who failed non-steroidal AI treatment, could potentially be more beneficial than EXE. Trial registration number UMIN000001841 PMID:23679192

  3. Use of MR-based trabecular bone microstructure analysis at the distal radius for osteoporosis diagnostics: a study in post-menopausal women with breast cancer and treated with aromatase inhibitor

    PubMed Central

    Baum, Thomas; Karampinos, Dimitrios C.; Seifert-Klauss, Vanadin; Pencheva, Tsvetelina D.; Jungmann, Pia M.; Rummeny, Ernst J.; Müller, Dirk; Bauer, Jan S.

    2016-01-01

    Summary Purpose Treatment with aromatase inhibitor (AI) is recommended for post-menopausal women with hormone-receptor positive breast cancer. However, AI therapy is known to induce bone loss leading to osteoporosis with an increased risk for fragility fractures. The purpose of this study was to investigate whether changes of magnetic resonance (MR)-based trabecular bone microstructure parameters as advanced imaging biomarker can already be detected in subjects with AI intake but still without evidence for osteoporosis according to dual energy X-ray absorptiometry (DXA)-based bone mineral density (BMD) measurements as current clinical gold standard. Methods Twenty-one postmenopausal women (62±6 years of age) with hormone-receptor positive breast cancer, ongoing treatment with aromatase inhibitor for 23±15 months, and no evidence for osteoporosis (current DXA T-score greater than −2.5) were recruited for this study. Eight young, healthy women (24±2 years of age) were included as controls. All subjects underwent 3 Tesla magnetic resonance imaging (MRI) of the distal radius to assess the trabecular bone microstructure. Results Trabecular bone microstructure parameters were not significantly (p>0.05) different between subjects with AI intake and controls, including apparent bone fraction (0.42±0.03 vs. 0.42±0.05), trabecular number (1.95±0.10 mm−1 vs 1.89±0.15 mm−1), trabecular separation (0.30±0.03 mm vs 0.31±0.06 mm), trabecular thickness (0.21±0.01 mm vs 0.22±0.02 mm), and fractal dimension (1.70±0.02 vs. 1.70±0.03). Conclusion These findings suggest that the initial deterioration of trabecular bone microstructure as measured by MRI and BMD loss as measured by DXA occur not sequentially but rather simultaneously. Thus, the use of MR-based trabecular bone microstructure assessment is limited as early diagnostic biomarker in this clinical setting. PMID:27252740

  4. Novel AChE Inhibitors for Sustainable Insecticide Resistance Management

    PubMed Central

    Alout, Haoues; Labbé, Pierrick; Berthomieu, Arnaud; Djogbénou, Luc; Leonetti, Jean-Paul; Fort, Philippe; Weill, Mylène

    2012-01-01

    Resistance to insecticides has become a critical issue in pest management and it is particularly chronic in the control of human disease vectors. The gravity of this situation is being exacerbated since there has not been a new insecticide class produced for over twenty years. Reasoned strategies have been developed to limit resistance spread but have proven difficult to implement in the field. Here we propose a new conceptual strategy based on inhibitors that preferentially target mosquitoes already resistant to a currently used insecticide. Application of such inhibitors in rotation with the insecticide against which resistance has been selected initially is expected to restore vector control efficacy and reduce the odds of neo-resistance. We validated this strategy by screening for inhibitors of the G119S mutated acetylcholinesterase-1 (AChE1), which mediates insensitivity to the widely used organophosphates (OP) and carbamates (CX) insecticides. PyrimidineTrione Furan-substituted (PTF) compounds came out as best hits, acting biochemically as reversible and competitive inhibitors of mosquito AChE1 and preferentially inhibiting the mutated form, insensitive to OP and CX. PTF application in bioassays preferentially killed OP-resistant Culex pipiens and Anopheles gambiae larvae as a consequence of AChE1 inhibition. Modeling the evolution of frequencies of wild type and OP-insensitive AChE1 alleles in PTF-treated populations using the selectivity parameters estimated from bioassays predicts a rapid rise in the wild type allele frequency. This study identifies the first compound class that preferentially targets OP-resistant mosquitoes, thus restoring OP-susceptibility, which validates a new prospect of sustainable insecticide resistance management. PMID:23056599

  5. Prediction of HIV-1 protease inhibitor resistance using a protein-inhibitor flexible docking approach.

    PubMed

    Jenwitheesuk, Ekachai; Samudrala, Ram

    2005-01-01

    Emergence of drug resistance remains one of the most challenging issues in the treatment of HIV-1 infection. Here we focus on resistance to HIV-1 protease inhibitors (PIs) at a molecular level, which can be analysed genotypically or phenotypically. Genotypic assays are based on the analysis of mutations associated with reduced drug susceptibility, but are problematic because of the numerous mutations and mutational patterns that confer drug resistance. Phenotypic resistance or susceptibility can be experimentally evaluated by measuring the amount of free drug bound to HIV-1 protease molecules, but this procedure is expensive and time-consuming. To overcome these problems, we have developed a docking protocol that takes protein-inhibitor flexibility into account to predict phenotypic drug resistance. For six FDA-approved Pls and a total of 1792 HIV-1 protease sequence mutants, we used a combination of inhibitor flexible docking and molecular dynamics (MD) simulations to calculate protein-inhibitor binding energies. Prediction results were expressed as fold changes of the calculated inhibitory constant (Ki), and the samples predicted to have fold-increase in calculated Ki above the fixed cut-off were defined as drug resistant. Our combined docking and MD protocol achieved accuracies ranging from 72-83% in predicting resistance/susceptibility for five of the six drugs evaluated. Evaluating the method only on samples where our predictions concurred with established knowledge-based methods resulted in increased accuracies of 83-94% for the six drugs. The results suggest that a physics-based approach, which is readily applicable to any novel PI and/or mutant, can be used judiciously with knowledge-based approaches that require experimental training data to devise accurate models of HIV-1 Pl resistance prediction.

  6. Molecular mechanisms of aromatase inhibition by new A, D-ring modified steroids.

    PubMed

    Cepa, Margarida; Correia-da-Silva, Georgina; Tavares da Silva, Elisiário J; Roleira, Fernanda M F; Hong, Yanyan; Chen, Shiuan; Teixeira, Natércia A

    2008-09-01

    A recent approach for treatment and prevention of estrogen-dependent breast cancer focuses on the inhibition of aromatase, the enzyme that catalyzes the final step of estrogen biosynthesis. Some synthetic steroids, such as formestane and exemestane, resembling the natural enzyme substrate androstenedione, revealed to be potent and useful aromatase inhibitors (AIs) and were approved for the treatment of estrogen-dependent breast cancer in postmenopausal women. Recently, we found that five newly synthesized steroids with chemical features in the A- and D-rings considered important for drug-receptor interaction efficiently inhibit aromatase derived from human placental microsomes. In this work, these steroids showed a similar pattern of anti-aromatase activity in several aromatase-expressing cell lines. 5alpha-androst-3-en-17-one and 3alpha,4alpha-epoxy-5alpha-androstan-17-one were revealed to be the most potent inhibitors. These compounds induced a time-dependent inhibition of aromatase, showing to be irreversible AIs. The specific interactions of these compounds with aromatase active sites were further demonstrated by site-directed mutagenesis studies and evaluated by computer-aided molecular modeling. Both compounds were able to suppress hormone-dependent proliferation of MCF-7aro cells in a dose-dependent manner. These findings are important for the elucidation of a structure-activity relationship on aromatase, which may help in the development of new AIs.

  7. Cross-resistance analysis of human immunodeficiency virus type 1 variants individually selected for resistance to five different protease inhibitors.

    PubMed Central

    Tisdale, M; Myers, R E; Maschera, B; Parry, N R; Oliver, N M; Blair, E D

    1995-01-01

    Human immunodeficiency virus type 1 (HIV-1) protease inhibitor-resistant variants, isolated on passage of HIV-1HXB2 in MT-4 cells with five different protease inhibitors, have been examined for cross-resistance to five inhibitors. The protease inhibitors studied were Ro 31-8959, A-77003, XM323, L-735,524, and VX-478. Resistant variants with two to four mutations within their protease sequence and 9- to 40-fold-decreased susceptibility were selected for all five inhibitors within six to eight passes in cell culture. Passage of a zidovudine-resistant mutant in Ro 31-8959 generated a dual reverse transcriptase- and protease-resistant virus. Variants were cloned directly into a modified pHXB2-D infectious clone for cross-resistance analysis. Although the resistant variants selected possessed different combinations of protease mutations for each inhibitor, many showed cross-resistance to the other inhibitors, and one showed cross-resistance to all five inhibitors. Interestingly, some mutants showed increased susceptibility to some inhibitors. Further HIV passage studies in the combined presence of two protease inhibitors demonstrated that in vitro it was possible to delay significantly selection of mutations producing resistance to one or both inhibitors. These studies indicate that there may be some rationale for combining different protease inhibitors as well as protease and reverse transcriptase inhibitors in HIV combination therapy. PMID:7486905

  8. Effect of the aromatase inhibitor CGS-16949A on pregnancy and secretion of progesterone, estradiol-17beta, prostaglandins E and F2alpha (PGE; PGF2alpha) and pregnancy specific protein B (PSPB) in 90-day ovariectomized pregnant ewes.

    PubMed

    Weems, Y S; Bridges, P J; LeaMaster, B R; Sasser, R G; Ching, L; Weems, C W

    2001-09-01

    The aromatase inhibitor CGS-16949A was used to determine whether CGS-16949A altered secretion of progesterone, estradiol-17beta, PGE (PGE1 + PGE2), PGF2alpha and PSPB. Ninety day pregnant ewes were ovariectomized and received vehicle, PGF2alpha, CGS-16949A or PGF2alpha+CGS-16949A. None of the ewes treated with PGF2alpha, CGS-16949A or PGF2alpha+CGS-16949A aborted (P > or = 0.05) during the 108-h experimental period. Treatment with CGS-16949A lowered (P < or = 0.05) progesterone in jugular venous plasma but concentrations of progesterone were not affected (P > or = 0.05) by treatment with PGF2alpha. Concentrations of estradiol-17beta and PSPB in jugular venous plasma and PGE in inferior vena cava plasma were decreased (P < or = 0.05) by treatment with CGS-16949A. Concentrations of PGF2alpha in inferior vena cava plasma were not affected (P > or = 0.05) by treatment with CGS-16949A. Decreases in estradiol-17beta occurred before decreases in PSPB, which was then followed by decreases in PGE (P < or = 0.05). It is concluded that these data support the hypothesis that estradiol-17beta regulates placental secretion of PSPB; PSPB regulates placental secretion of PGE; and PGE regulates placental secretion of progesterone during mid-pregnancy in ewes.

  9. Targeting mitochondrial biogenesis to overcome drug resistance to MAPK inhibitors

    PubMed Central

    Zhang, Gao; Frederick, Dennie T.; Wu, Lawrence; Wei, Zhi; Krepler, Clemens; Srinivasan, Satish; Chae, Young Chan; Xu, Xiaowei; Choi, Harry; Dimwamwa, Elaida; Shannan, Batool; Basu, Devraj; Zhang, Dongmei; Guha, Manti; Xiao, Min; Randell, Sergio; Sproesser, Katrin; Xu, Wei; Liu, Jephrey; Karakousis, Giorgos C.; Schuchter, Lynn M.; Gangadhar, Tara C.; Amaravadi, Ravi K.; Gu, Mengnan; Xu, Caiyue; Ghosh, Abheek; Xu, Weiting; Tian, Tian; Zhang, Jie; Zha, Shijie; Brafford, Patricia; Weeraratna, Ashani; Davies, Michael A.; Wargo, Jennifer A.; Avadhani, Narayan G.; Lu, Yiling; Mills, Gordon B.; Altieri, Dario C.; Flaherty, Keith T.

    2016-01-01

    Targeting multiple components of the MAPK pathway can prolong the survival of patients with BRAFV600E melanoma. This approach is not curative, as some BRAF-mutated melanoma cells are intrinsically resistant to MAPK inhibitors (MAPKi). At the systemic level, our knowledge of how signaling pathways underlie drug resistance needs to be further expanded. Here, we have shown that intrinsically resistant BRAF-mutated melanoma cells with a low basal level of mitochondrial biogenesis depend on this process to survive MAPKi. Intrinsically resistant cells exploited an integrated stress response, exhibited an increase in mitochondrial DNA content, and required oxidative phosphorylation to meet their bioenergetic needs. We determined that intrinsically resistant cells rely on the genes encoding TFAM, which controls mitochondrial genome replication and transcription, and TRAP1, which regulates mitochondrial protein folding. Therefore, we targeted mitochondrial biogenesis with a mitochondrium-targeted, small-molecule HSP90 inhibitor (Gamitrinib), which eradicated intrinsically resistant cells and augmented the efficacy of MAPKi by inducing mitochondrial dysfunction and inhibiting tumor bioenergetics. A subset of tumor biopsies from patients with disease progression despite MAPKi treatment showed increased mitochondrial biogenesis and tumor bioenergetics. A subset of acquired drug-resistant melanoma cell lines was sensitive to Gamitrinib. Our study establishes mitochondrial biogenesis, coupled with aberrant tumor bioenergetics, as a potential therapy escape mechanism and paves the way for a rationale-based combinatorial strategy to improve the efficacy of MAPKi. PMID:27043285

  10. Caught in a Network: Recovery from Aromatase Inhibition

    EPA Science Inventory

    Fadrozole is an inhibitor of aromatase, an enzyme critical to estrogen synthesis. We exposed female fathead minnows (Pimephales promelas, FHM) to 0 or 30 ug/L fadrozole for 8 days, and fish were then held in clean water for 8 extra days. We analyzed ex vivo steroid production, pl...

  11. Reduced methadone clearance during aromatase inhibition.

    PubMed

    Lu, Wenjie Jessie; Thong, Nancy; Flockhart, David A

    2012-08-01

    Methadone is increasingly used in pain management and is a cornerstone in the treatment of opiate withdrawal. It is subject to highly variable clearance among patients. The complete metabolic disposition of methadone is likely to involve a number of enzymes, including specifically CYP2B6. Previous studies in vitro suggest that metabolism by aromatase may also contribute. Single-dose methadone pharmacokinetics (2 mg, intravenous) were studied in 15 healthy postmenopausal women in the presence and absence of a potent aromatase inhibitor, letrozole. A sequential design was used, involving a control period followed by treatment with letrozole (2.5 mg/d, 11 days), in which each subject served as her own control. On average, letrozole treatment reduced methadone systemic clearance by 22% (P = 0.001), increased methadone AUC by 23% (P = 0.007), and increased elimination half-life by 21% (P = 0.042). The plasma parent-to-metabolite ratio also increased (P = 0.009), and there was a linear relationship (R2 = 0.74) between change in this plasma ratio and change in methadone AUC0-∞. In contrast, there was no such association with change in apparent urinary methadone clearance. Letrozole did not change methadone distribution half-life or its volume of distribution. Overall, these data demonstrate a significant decrease in methadone clearance during coadministration of letrozole, consistent with decreased metabolism brought about by aromatase inhibition. An involvement of aromatase in the disposition of methadone may help explain the difficulty in methadone dosing and suggests a broader role for this catalyst of endogenous steroid metabolism in xenobiotic drug disposition.

  12. Non-target-site resistance to ALS inhibitors in waterhemp (Amaranthus tuberculatus)

    Technology Transfer Automated Retrieval System (TEKTRAN)

    A waterhemp population (MCR) previously characterized as resistant to 4-hyroxyphenylpyruvate dioxygenase (HPPD) and photosystem II (PSII) inhibitors was found to have two different resistance responses to acetolactate synthase (ALS) inhibitors. Plants from the MCR population exhibiting high resistan...

  13. History of aromatase: saga of an important biological mediator and therapeutic target.

    PubMed

    Santen, R J; Brodie, H; Simpson, E R; Siiteri, P K; Brodie, A

    2009-06-01

    Aromatase is the enzyme that catalyzes the conversion of androgens to estrogens. Initial studies of its enzymatic activity and function took place in an environment focused on estrogen as a component of the birth control pill. At an early stage, investigators recognized that inhibition of this enzyme could have major practical applications for treatment of hormone-dependent breast cancer, alterations of ovarian and endometrial function, and treatment of benign disorders such as gynecomastia. Two general approaches ultimately led to the development of potent and selective aromatase inhibitors. One targeted the enzyme using analogs of natural steroidal substrates to work out the relationships between structure and function. The other approach initially sought to block adrenal function as a treatment for breast cancer but led to the serendipitous finding that a nonsteroidal P450 steroidogenesis inhibitor, aminoglutethimide, served as a potent but nonselective aromatase inhibitor. Proof of the therapeutic concept of aromatase inhibition involved a variety of studies with aminoglutethimide and the selective steroidal inhibitor, formestane. The requirement for even more potent and selective inhibitors led to intensive molecular studies to identify the structure of aromatase, to development of high-sensitivity estrogen assays, and to "mega" clinical trials of the third-generation aromatase inhibitors, letrozole, anastrozole, and exemestane, which are now in clinical use in breast cancer. During these studies, unexpected findings led investigators to appreciate the important role of estrogens in males as well as in females and in multiple organs, particularly the bone and brain. These studies identified the important regulatory properties of aromatase acting in an autocrine, paracrine, intracrine, neurocrine, and juxtacrine fashion and the organ-specific enhancers and promoters controlling its transcription. The saga of these studies of aromatase and the ultimate

  14. Biochemical and computational insights into the anti-aromatase activity of natural catechol estrogens.

    PubMed

    Neves, Marco A C; Dinis, Teresa C P; Colombo, Giorgio; Luisa Sá E Melo, M

    2008-05-01

    High levels of endogenous estrogens are associated with increased risks of breast cancer. Estrogen levels are mainly increased by the activity of the aromatase enzyme and reduced by oxidative/conjugative metabolic pathways. In this paper, we demonstrate for the first time that catechol estrogen metabolites are potent aromatase inhibitors, thus establishing a link between aromatase activity and the processes involved in estrogen metabolism. In particular, the anti-aromatase activity of a set of natural hydroxyl and methoxyl estrogen metabolites was investigated using biochemical methods and subsequently compared with the anti-aromatase potency of estradiol and two reference aromatase inhibitors. Catechol estrogens proved to be strong inhibitors with an anti-aromatase potency two orders of magnitude higher than estradiol. A competitive inhibition mechanism was found for the most potent molecule, 2-hydroxyestradiol (2-OHE(2)) and a rational model identifying the interaction determinants of the metabolites with the enzyme is proposed based on ab initio quantum-mechanical calculations. A strong relationship between activity and electrostatic properties was found for catechol estrogens. Moreover, our results suggest that natural catechol estrogens may be involved in the control mechanisms of estrogen production.

  15. Glyceollin I reverses epithelial to mesenchymal transition in letrozole resistance

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Although aromatase inhibitors, such as letrozole; are standard endocrine therapy for postmenopausal women with early stage metastatic estrogen-dependent breast cancer, the major limitation in managing this disease is the development of drug resistance; therefore, a better understanding of this proce...

  16. Nicotine Blocks Brain Estrogen Synthase (Aromatase): In Vivo Positron Emission Tomography Studies in Female Baboons

    SciTech Connect

    Biegon, A.; Biegon, A.; Kim, S.-W.; Logan, J.; Hooker, J.M.; Muench, L.; Fowler, J.S.

    2010-01-12

    Cigarette smoking and nicotine have complex effects on human physiology and behavior, including some effects similar to those elicited by inhibition of aromatase, the last enzyme in estrogen biosynthesis. We report the first in vivo primate study to determine whether there is a direct effect of nicotine administration on brain aromatase. Brain aromatase availability was examined with positron emission tomography and the selective aromatase inhibitor [{sup 11}C]vorozole in six baboons before and after exposure to IV nicotine at .015 and .03 mg/kg. Nicotine administration produced significant, dose-dependent reductions in [{sup 11}C]vorozole binding. The amygdala and preoptic area showed the largest reductions. Plasma levels of nicotine and its major metabolite cotinine were similar to those found in cigarette smokers. Nicotine interacts in vivo with primate brain aromatase in regions involved in mood, aggression, and sexual behavior.

  17. Molecular pharmacology of aromatase and its regulation by endogenous and exogenous agents.

    PubMed

    Brueggemeier, R W; Richards, J A; Joomprabutra, S; Bhat, A S; Whetstone, J L

    2001-12-01

    focus on diversifying the benzopyranone scaffold and utilizing combinatorial chemistry approaches to construct small benzopyranone libraries as potential aromatase inhibitors.

  18. Resistance to HER2-directed antibodies and tyrosine kinase inhibitors

    PubMed Central

    Garrett, Joan T

    2011-01-01

    The antibody trastuzumab and the tyrosine kinase inhibitor lapatinib are approved by the FDA for the treatment of HER2-overexpressing breast cancer. These anti-HER2 drugs are changing the natural history of HER2-overexpressing breast cancer. However, therapeutic resistance to trastuzumab or lapatinib, as either single-agents or in combination with chemotherapy in the metastatic setting, typically occurs within months of starting therapy. Several mechanisms of trastuzumab-resistance have been reported that include signaling from other HER receptors, signaling from receptor tyrosine kinases (RTKs) outside of the HER (ErbB) family, increased phosphatidylinositol-3-kinase signaling, and the presence of truncated forms of HER2. Mechanisms of resistance to lapatinib also point to increased phosphatidylinositol 3-kinase signaling as well as derepression/activation of compensatory survival pathways. In this review, we discuss how these models and mechanisms enhance our understanding of the clinical resistance to HER2-directed therapies. PMID:21307659

  19. The STAT5 inhibitor pimozide decreases survival of chronic myelogenous leukemia cells resistant to kinase inhibitors

    PubMed Central

    Nelson, Erik A.; Walker, Sarah R.; Weisberg, Ellen; Bar-Natan, Michal; Barrett, Rosemary; Gashin, Laurie B.; Terrell, Shariya; Klitgaard, Josephine L.; Santo, Loredana; Addorio, Martha R.; Ebert, Benjamin L.; Griffin, James D.

    2011-01-01

    The transcription factor STAT5 is an essential mediator of the pathogenesis of chronic myelogenous leukemia (CML). In CML, the BCR/ABL fusion kinase causes the constitutive activation of STAT5, thereby driving the expression of genes promoting survival. BCR/ABL kinase inhibitors have become the mainstay of therapy for CML, although CML cells can develop resistance through mutations in BCR/ABL. To overcome this problem, we used a cell-based screen to identify drugs that inhibit STAT-dependent gene expression. Using this approach, we identified the psychotropic drug pimozide as a STAT5 inhibitor. Pimozide decreases STAT5 tyrosine phosphorylation, although it does not inhibit BCR/ABL or other tyrosine kinases. Furthermore, pimozide decreases the expression of STAT5 target genes and induces cell cycle arrest and apoptosis in CML cell lines. Pimozide also selectively inhibits colony formation of CD34+ bone marrow cells from CML patients. Importantly, pimozide induces similar effects in the presence of the T315I BCR/ABL mutation that renders the kinase resistant to presently available inhibitors. Simultaneously inhibiting STAT5 with pimozide and the kinase inhibitors imatinib or nilotinib shows enhanced effects in inhibiting STAT5 phosphorylation and in inducing apoptosis. Thus, targeting STAT5 may be an effective strategy for the treatment of CML and other myeloproliferative diseases. PMID:21233313

  20. Aromatase inhibitors alone or sequentially combined with tamoxifen in postmenopausal early breast cancer compared with tamoxifen or placebo - Meta-analyses on efficacy and adverse events based on randomized clinical trials.

    PubMed

    Rydén, Lisa; Heibert Arnlind, Marianne; Vitols, Sigurd; Höistad, Malin; Ahlgren, Johan

    2016-04-01

    Tamoxifen (TAM) and aromatase inhibitors (AI) are adjuvant therapy options for postmenopausal women with estrogen receptor positive (ER+) breast cancer. This systematic review of seven randomized controlled studies comparing TAM and AI, and one study comparing extended therapy with an AI with placebo after about 5 years of tamoxifen, aims to assess long-term clinical efficacy and adverse events. The literature review was performed according to the principles of the Cochrane Collaboration. The search included common databases up to 2013-01-14. Studies of high or moderate quality were used for grading of evidence. Revman™ software was utilized for meta-analyses of published data. Disease free survival (DFS) and overall survival (OS) were improved with AI monotherapy compared to TAM with high and moderate quality of evidence respectively. Sequenced therapy with AI → TAM (or vice versa) improved DFS compared with TAM with moderate quality of evidence, but did not improve OS (low quality of evidence). However, if only studies on sequenced AI therapy with randomization before endocrine therapy were considered, no improvement of DFS could be found. Fractures are more frequently associated with AI whereas the risk of endometrial cancer and venous thromboembolism are higher with TAM. For cardiovascular events no difference was found between AI (mono- or sequenced therapy) and TAM, whereas sequenced therapy compared with AI had lower risk of cardiovascular events (moderate level of evidence). AIs are superior to TAM as adjuvant hormonal therapy for postmenopausal ER-positive breast cancer. TAM can be considered for individual patients due to the different toxicity profile compared with AI. Cardiovascular events related to AI treatment deserve further attention.

  1. Mechanisms of resistance to EGFR tyrosine kinase inhibitors

    PubMed Central

    Huang, Lihua; Fu, Liwu

    2015-01-01

    Since the discovery that non-small cell lung cancer (NSCLC) is driven by epidermal growth factor receptor (EGFR) mutations, the EGFR tyrosine kinase inhibitors (EGFR-TKIs, e.g., gefitinib and elrotinib) have been effectively used for clinical treatment. However, patients eventually develop drug resistance. Resistance to EGFR-TKIs is inevitable due to various mechanisms, such as the secondary mutation (T790M), activation of alternative pathways (c-Met, HGF, AXL), aberrance of the downstream pathways (K-RAS mutations, loss of PTEN), impairment of the EGFR-TKIs-mediated apoptosis pathway (BCL2-like 11/BIM deletion polymorphism), histologic transformation, ATP binding cassette (ABC) transporter effusion, etc. Here we review and summarize the known resistant mechanisms to EGFR-TKIs and provide potential targets for development of new therapeutic strategies. PMID:26579470

  2. Genetics Home Reference: aromatase deficiency

    MedlinePlus

    ... development, aromatase converts androgens to estrogens in the placenta, which is the link between the mother's blood supply and the fetus. This conversion in the placenta prevents androgens from directing sexual development in female ...

  3. Competitive product inhibition of aromatase by natural estrogens.

    PubMed

    Shimizu, Y; Yarborough, C; Osawa, Y

    1993-03-01

    In order to better understand the function of aromatase, we carried out kinetic analyses to assess the ability of natural estrogens, estrone (E1), estradiol (E2), 16 alpha-OHE1, and estriol (E3), to inhibit aromatization. Human placental microsomes (50 micrograms protein) were incubated for 5 min at 37 degrees C with [1 beta-3H]testosterone (1.24 x 10(3) dpm 3H/ng, 35-150 nM) or [1 beta-3H,4-14C]androstenedione (3.05 x 10(3) dpm 3H/ng, 3H/14C = 19.3, 7-65 nM) as substrate in the presence of NADPH, with and without natural estrogens as putative inhibitors. Aromatase activity was assessed by tritium released to water from the 1 beta-position of the substrates. Natural estrogens showed competitive product inhibition against androgen aromatization. The Ki of E1, E2, 16 alpha-OHE1, and E3 for testosterone aromatization was 1.5, 2.2, 95, and 162 microM, respectively, where the Km of aromatase was 61.8 +/- 2.0 nM (n = 5) for testosterone. The Ki of E1, E2, 16 alpha-OHE1, and E3 for androstenedione aromatization was 10.6, 5.5, 252, and 1182 microM, respectively, where the Km of aromatase was 35.4 +/- 4.1 nM (n = 4) for androstenedione. These results show that estrogen inhibit the process of androgen aromatization and indicate that natural estrogens regulate their own synthesis by the product inhibition mechanism in vivo. Since natural estrogen binds to the active site of human placental aromatase P-450 complex as competitive inhibitors, natural estrogens might be further metabolized by aromatase. This suggests that human placental estrogen 2-hydroxylase activity is catalyzed by the active site of aromatase cytochrome P-450 and also agrees with the fact that the level of catecholestrogens in maternal plasma increases during pregnancy. The relative affinities and concentration of androgens and estrogens would control estrogen and catecholestrogen biosynthesis by aromatase.

  4. Identification of cyclohexanone derivatives that act as catalytic inhibitors of topoisomerase I: effects on tamoxifen-resistant MCF-7 cancer cells.

    PubMed

    Leung, Euphemia; Rewcastle, Gordon W; Joseph, Wayne R; Rosengren, Rhonda J; Larsen, Lesley; Baguley, Bruce C

    2012-12-01

    Breast cancer is commonly treated with anti-estrogens or aromatase inhibitors, but resistant disease eventually develops and new therapies for such resistance are of great interest. We have previously isolated several tamoxifen-resistant variant sub-lines of the MCF-7 breast cancer cell line and provided evidence that they arose from expansion of pre-existing minor populations. We have searched for therapeutic agents that exhibit selective growth inhibition of the resistant lines and here investigate 2,6-bis(pyridin-3-ylmethylene)-cyclohexanone (RL90) and 2,6-bis(pyridin-4-ylmethylene)-cyclohexanone (RL91). We found that two of the tamoxifen-resistant sub-lines (TamR3 and TamC3) unexpectedly showed increased sensitivity to RL90 and RL91. We utilized growth inhibition assays, flow cytometry and immunoblotting to establish a mechanistic basis for their action. Treated sensitive cells showed S-phase selective DNA damage, as detected by histone H2AX phosphorylation. Cellular responses were similar to those induced by the topoisomerase I poison camptothecin. Although IC(50) values of camptothecin, RL90, RL91 were correlated, studies with purified mammalian topoisomerase I suggested that RL90 and RL91 differed from camptothecin by acting as catalytic topoisomerase I inhibitors. These drugs provide a platform for the further development of DNA damaging drugs that have selective effects on tamoxifen resistant breast cancer cells. The results also raise the question of whether clinical topoisomerase I poisons such as irinotecan and topotecan might be active in the treatment of some types of tamoxifen-resistant cancer.

  5. Avibactam and Inhibitor-Resistant SHV β-Lactamases

    PubMed Central

    Winkler, Marisa L.; Papp-Wallace, Krisztina M.; Taracila, Magdalena A.

    2015-01-01

    β-Lactamase enzymes (EC 3.5.2.6) are a significant threat to the continued use of β-lactam antibiotics to treat infections. A novel non-β-lactam β-lactamase inhibitor with activity against many class A and C and some class D β-lactamase variants, avibactam, is now available in the clinic in partnership with ceftazidime. Here, we explored the activity of avibactam against a variety of characterized isogenic laboratory constructs of β-lactamase inhibitor-resistant variants of the class A enzyme SHV (M69I/L/V, S130G, K234R, R244S, and N276D). We discovered that the S130G variant of SHV-1 shows the most significant resistance to inhibition by avibactam, based on both microbiological and biochemical characterizations. Using a constant concentration of 4 mg/liter of avibactam as a β-lactamase inhibitor in combination with ampicillin, the MIC increased from 1 mg/liter for blaSHV-1 to 256 mg/liter for blaSHV S130G expressed in Escherichia coli DH10B. At steady state, the k2/K value of the S130G variant when inactivated by avibactam was 1.3 M−1 s−1, versus 60,300 M−1 s−1 for the SHV-1 β-lactamase. Under timed inactivation conditions, we found that an approximately 1,700-fold-higher avibactam concentration was required to inhibit SHV S130G than the concentration that inhibited SHV-1. Molecular modeling suggested that the positioning of amino acids in the active site of SHV may result in an alternative pathway of inactivation when complexed with avibactam, compared to the structure of CTX-M-15–avibactam, and that S130 plays a role in the acylation of avibactam as a general acid/base. In addition, S130 may play a role in recyclization. As a result, we advance that the lack of a hydroxyl group at position 130 in the S130G variant of SHV-1 substantially slows carbamylation of the β-lactamase by avibactam by (i) removing an important proton acceptor and donator in catalysis and (ii) decreasing the number of H bonds. In addition, recyclization is most likely

  6. Regulation of aromatase expression in breast cancer tissue.

    PubMed

    Bulun, S E; Lin, Z; Zhao, H; Lu, M; Amin, S; Reierstad, S; Chen, D

    2009-02-01

    Epithelial-stromal interactions play key roles for aromatase expression and estrogen production in breast cancer tissue. Upregulated aromatase expression in breast fibroblasts increases the tissue concentration of estradiol (E2), which then activates a large number of carcinogenic genes via estrogen receptor-alpha (ERalpha) in malignant epithelial cells. This clinically pertains, since aromatase inhibitors (AIs) are the most effective hormonal treatment of ERalpha-positive breast tumors. A single gene encodes aromatase, the key enzyme in estrogen biosynthesis, the inhibition of which by an AI effectively eliminates E2 production. Since alternative promoters regulated by distinct signaling pathways control aromatase expression, it is possible to target these pathways and inhibit estrogen production in a tissue-selective fashion. We and others previously found that the majority of estrogen production in breast cancer tissue was accounted for by the aberrant activation of the proximal promoter I.3/II region. PGE(2) that is secreted in large amounts by malignant breast epithelial cells is the most potent known natural inducer of this promoter region in breast adipose fibroblasts. Signaling effectors/transcriptional regulators that mediate PGE(2) action include the activator pathways p38/CREB-ATF and JNK/jun and the inhibitory factor BRCA1 in breast adipose fibroblasts. Selective inhibition of this promoter region may treat breast cancer while permitting aromatase expression via alternative promoters in the brain and bone and thus obviate the key side effects of the current AIs. The signaling pathways that mediate the regulation of the promoter I.3/II region in undifferentiated fibroblasts in malignant breast tumors are reviewed.

  7. Drug Repurposing Identifies Inhibitors of Oseltamivir-Resistant Influenza Viruses.

    PubMed

    Bao, Ju; Marathe, Bindumadhav; Govorkova, Elena A; Zheng, Jie J

    2016-03-01

    The neuraminidase (NA) inhibitor, oseltamivir, is a widely used anti-influenza drug. However, oseltamivir-resistant H1N1 influenza viruses carrying the H275Y NA mutation spontaneously emerged as a result of natural genetic drift and drug treatment. Because H275Y and other potential mutations may generate a future pandemic influenza strain that is oseltamivir-resistant, alternative therapy options are needed. Herein, we show that a structure-based computational method can be used to identify existing drugs that inhibit resistant viruses, thereby providing a first line of pharmaceutical defense against this possible scenario. We identified two drugs, nalidixic acid and dorzolamide, that potently inhibit the NA activity of oseltamivir-resistant H1N1 viruses with the H275Y NA mutation at very low concentrations, but have no effect on wild-type H1N1 NA even at a much higher concentration, suggesting that the oseltamivir-resistance mutation itself caused susceptibility to these drugs.

  8. Suppression of aromatase activity in vitro by PCBs 28 and 105 and Aroclor 1221.

    PubMed

    Woodhouse, Amanda J; Cooke, Gerard M

    2004-08-30

    The effects of polychlorinated biphenyls (PCBs) on human cytochrome P450 aromatase activity in vitro were investigated using a commercially available microsomal fraction obtained from baculovirus infected insects that had been transfected with the human CYP19 gene and cytochrome P450 reductase. The assay measured the conversion of tritiated testosterone to estradiol in Tris buffer at pH 7.4. When aroclors, commercial preparations of PCBs, were added to aromatase assays at a 10 microM concentration, Aroclor 1221 caused a reduction in the aromatase activity, whereas other aroclors (1016, 1232, 1242, 1248, 1254, 1260, 5432, 5442 and 5460) were without effect. Further investigation of the effect of Aroclor 1221 on aromatase activity showed that the inhibition was dose dependent. When a reconstituted mixture (RM) of PCBs that represented the congeneric content of human milk was investigated, no inhibition of aromatase activity at the maximum treatment of 15.0 microM was observed. None of the congeners present in the reconstituted mixture, except PCB 28 and 105, affected P450 arom activity. PCB 28 showed a statistically significant inhibition of aromatase activity (P<0.05) at 1.5 and 15 microM and a significant inhibition of aromatase activity by PCB 105 was also observed, but only at 15 microM. In three separate kinetic analyses the Km(app) for aromatase was 64, 89 and 69 nM (mean 74 nM). In addition, PCB 28 resulted in an increase in the Km(app) without a significant effect on Vmax(app), suggesting competitive inhibition by this congener. This conclusion was supported by slope (Km(app)/Vmax(app) versus [inhibitor]) and intercept (1/Vmax(app) versus [inhibitor]) replots. The slope replots gave Ki(app) values for PCB 28 of 0.9, 1.3 and 2.0 microM (mean 1.4 microM), whereas intercept replots were almost horizontal. Thus, PCB 28 is a competitive inhibitor of aromatase with a Ki(app) value approximately 20-fold the Km(app) value. Based on these studies, we conclude that

  9. Overcoming Resistance to Inhibitors of the Akt Protein Kinase by Modulation of the Pim Kinase Pathway

    DTIC Science & Technology

    2014-10-01

    kinase . This grant proposal will explore the resistance to small molecule AKT protein kinase inhibitors mediated by the... molecule AKT protein kinase inhibitors is potentially mediated by the Pim-1 protein kinase , and that unique Pim protein kinase inhibitors that can in...application is essential for the development of this combined chemotherapeutic strategy. 15. SUBJECT TERMS Small Molecule AKT Inhibitors ,

  10. Studies on pyrrolopyrimidines as selective inhibitors of multidrug-resistance-associated protein in multidrug resistance.

    PubMed

    Wang, Shouming; Folkes, Adrian; Chuckowree, Irina; Cockcroft, Xiaoling; Sohal, Sukhjit; Miller, Warren; Milton, John; Wren, Stephen P; Vicker, Nigel; Depledge, Paul; Scott, John; Smith, Lyndsay; Jones, Hazel; Mistry, Prakash; Faint, Richard; Thompson, Deanne; Cocks, Simon

    2004-03-11

    Multidrug resistance mediated by P-glycoprotein (Pgp) or multidrug-resistance-associated protein (MRP) remains a major obstacle for successful treatment of cancer. Inhibition of Pgp and MRP transport is important for high efficacy of anticancer drugs. While several Pgp inhibitors have entered clinical trials, the development of specific MRP1 inhibitors is still in its infancy. In our screening program, we have identified a pyrrolopyrimidine (4) as a novel and selective MRP1 inhibitor. Subsequent SAR work on the 4-position of the template revealed the phenethylpiperazine side chain as a potent replacement of the benzylthio group of the lead molecule. Introduction of groups at the 2-position seems to have no detrimental effect on activity. Modifications to the nitrile group at the 7-position resulted in the identification of analogues with groups, such as amides, with superior pharmacokinetic profiles. In vivo efficacy has been demonstrated by xenograft studies on selected compounds.

  11. Aromatase inhibition by synthetic lactones and flavonoids in human placental microsomes and breast fibroblasts - A comparative study

    SciTech Connect

    Meeuwen, J.A. van Nijmeijer, S.; Mutarapat, T.; Ruchirawat, S.; Jong, P.C. de; Piersma, A.H.; Berg, M. van den

    2008-05-01

    Interference of exogenous chemicals with the aromatase enzyme can be useful as a tool to identify chemicals that could act either chemopreventive for hormone-dependent cancer or adverse endocrine disruptive. Aromatase is the key enzyme in the biosynthesis of steroids, as it converts androgens to estrogens. Certain flavonoids, plant derived chemicals, are known catalytic aromatase inhibitors. Various systems are in use to test aromatase inhibitory properties of compounds. Commonly used are microsomes derived from ovary or placental tissue characterized by high aromatase activity. To a lesser extent whole cell systems are used and specifically cell systems that are potential target tissue in breast cancer development. In this study aromatase inhibitory properties of fadrozole, 8-prenylnaringenin and a synthetic lactone (TM-7) were determined in human placental microsomes and in human primary breast fibroblasts. In addition, apigenin, chrysin, naringenin and two synthetic lactones (TM-8 and TM-9) were tested in human microsomes only. Comparison of the aromatase inhibitory potencies of these compounds between the two test systems showed that the measurement of aromatase inhibition in human placental microsomes is a good predictor of aromatase inhibition in human breast fibroblasts.

  12. Aromatase inhibition by synthetic lactones and flavonoids in human placental microsomes and breast fibroblasts--a comparative study.

    PubMed

    van Meeuwen, J A; Nijmeijer, S; Mutarapat, T; Ruchirawat, S; de Jong, P C; Piersma, A H; van den Berg, M

    2008-05-01

    Interference of exogenous chemicals with the aromatase enzyme can be useful as a tool to identify chemicals that could act either chemopreventive for hormone-dependent cancer or adverse endocrine disruptive. Aromatase is the key enzyme in the biosynthesis of steroids, as it converts androgens to estrogens. Certain flavonoids, plant derived chemicals, are known catalytic aromatase inhibitors. Various systems are in use to test aromatase inhibitory properties of compounds. Commonly used are microsomes derived from ovary or placental tissue characterized by high aromatase activity. To a lesser extent whole cell systems are used and specifically cell systems that are potential target tissue in breast cancer development. In this study aromatase inhibitory properties of fadrozole, 8-prenylnaringenin and a synthetic lactone (TM-7) were determined in human placental microsomes and in human primary breast fibroblasts. In addition, apigenin, chrysin, naringenin and two synthetic lactones (TM-8 and TM-9) were tested in human microsomes only. Comparison of the aromatase inhibitory potencies of these compounds between the two test systems showed that the measurement of aromatase inhibition in human placental microsomes is a good predictor of aromatase inhibition in human breast fibroblasts.

  13. Role of P-450 aromatase in sex determination of the diamondback terrapin, Malaclemys terrapin.

    PubMed

    Jeyasuria, P; Roosenburg, W M; Place, A R

    1994-09-15

    Sex determination in the diamondback terrapin, Malaclemys terrapin, is temperature-dependent. Eggs incubated at 31 degrees C, and above, hatch in approximately 45 days as females. Eggs incubated below 27 degrees C hatch in about 60 days as males. Sex is not reversible after hatching. Nest temperatures in the wild can be as low as 20 degrees C and as high as 37 degrees C with as much as a 10 degrees C diel cycle. The shortest incubation time measured in nature was 56 days and the longest approaching 120 days. Nests in our study site produced predominantly (> 95%) male hatchlings. Treatment of developing embryos with estrogen produces females at male producing temperatures while treatment with fadrozole (a nonsteroidal aromatase inhibitor) induces partial male-like gonads. Treatment with a steroidal aromatase inhibitor (4-hydroxyandrostenedione, 4-OHA) had no effect on sex determination. Both fadrozole and 4-OHA are potent competitive inhibitors (Ki approximately 40-50 nM) for terrapin in vitro aromatase activity. These findings are consistent with aromatase expression being a key step in sex determination of terrapins. We have cloned a partial single copy P-450 aromatase from the terrapin using a cDNA library constructed from ovarian mRNA. This partial clone is highly homologous to other vertebrate aromatases.

  14. Genetics Home Reference: aromatase excess syndrome

    MedlinePlus

    ... Sources for This Page Fukami M, Shozu M, Ogata T. Molecular bases and phenotypic determinants of aromatase ... T, Nishigaki T, Yokoya S, Binder G, Horikawa R, Ogata T. Aromatase excess syndrome: identification of cryptic duplications ...

  15. High Proteolytic Resistance of Spider-Derived Inhibitor Cystine Knots

    PubMed Central

    Kikuchi, Kyoko; Sugiura, Mika; Kimura, Tadashi

    2015-01-01

    Proteolytic stability in gastrointestinal tract and blood plasma is the major obstacle for oral peptide drug development. Inhibitor cystine knots (ICKs) are linear cystine knot peptides which have multifunctional properties and could become promising drug scaffolds. ProTx-I, ProTx-II, GTx1-15, and GsMTx-4 were spider-derived ICKs and incubated with pepsin, trypsin, chymotrypsin, and elastase in physiological conditions to find that all tested peptides were resistant to pepsin, and ProTx-II, GsMTx-4, and GTx1-15 showed resistance to all tested proteases. Also, no ProTx-II degradation was observed in rat blood plasma for 24 hours in vitro and ProTx-II concentration in circulation decreased to half in 40 min, indicating absolute stability in plasma and fast clearance from the system. So far, linear peptides are generally thought to be unsuitable in vivo, but all tested ICKs were not degraded by pepsin and stomach could be selected for the alternative site of drug absorption for fast onset of the drug action. Since spider ICKs are selective inhibitors of various ion channels which are related to the pathology of many diseases, engineered ICKs will make a novel class of peptide medicines which can treat variety of bothering symptoms. PMID:26843868

  16. PAAn-1b and PAAn-E: two phosphorothioate antisense oligodeoxynucleotides inhibit human aromatase gene expression.

    PubMed

    Auvray, P; Sourdaine, P; Séralini, G E

    1998-12-09

    Estrogen-dependent diseases, especially breast cancers, are frequently treated with aromatase inhibitors. Another more recent strategy is the antisense technology. In this study, after predicting aromatase mRNA secondary structure, we describe the design, the efficiency, and the toxicity of two antisense phosphorothioate oligodeoxynucleotides (PAAn-1b and PAAn-E) directed toward aromatase mRNA. Indeed, 2 microM PAAn-1b and PAAn-E encapsulated with 54 microM polyethylenimine inhibit aromatase activity by 71 and 79%, respectively, in transfected 293 cells, with IC50 values of 0.2 and 0.6 microM. The mechanism of inhibition appears to be specific after using sense and scramble oligodeoxynucleotides as controls and largely decreases aromatase mRNA and protein amounts. Moreover, PAAn-1b and PAAn-E are not cytotoxic for 293 cells. This study finally provides a new strategy for aromatase inhibition. It offers new tools for studying aromatase gene expression and its role in cancer for instance, and this could be of help for the therapy of estrogen-dependent diseases.

  17. Quantitative AOP-based predictions for two aromatase ...

    EPA Pesticide Factsheets

    The adverse outcome pathway (AOP) framework can be used to support the use of mechanistic toxicology data as a basis for risk assessment. For certain risk contexts this includes defining, quantitative linkages between the molecular initiating event (MIE) and subsequent key events (KEs) within an AOP. One AOP for which strong, quantitative linkages have been established is aromatase inhibition leading to reproductive dysfunction in fish. A series of computational models have been linked to develop a quantitative AOP (Q-AOP). A measure of aromatase inhibition is used as the model input to estimate circulating plasma estradiol (E2) concentration and resultant circulating plasma vitellogenin (VTG) concentration. To evaluate model predictions, two aromatase inhibitors, letrozole and epoxiconazole, were selected based upon their relative aromatase inhibition potency in US EPA ToxCast assays. Reproductively mature female fathead minnows (Pimephales promelas) were exposed to varying concentrations of either letrozole (0.5, 7.5, 25, 75, 250 µg/L) or epoxiconazole (8, 25, 80, 250, 800 µg/L) in 24h flow through exposures. One additional consideration for model predictions was bioaccumulation of exposure chemicals and resultant circulating plasma concentration. To identify this, plasma from exposed minnows was extracted by supported liquid extraction (SLE) and concentrations of letrozole or epoxiconazole determined by LC-MS/MS. Plasma bioaccumulation factors (BAFplasma)

  18. A three-dimensional model of CYP19 aromatase for structure-based drug design.

    PubMed

    Karkola, Sampo; Höltje, Hans-Dieter; Wähälä, Kristiina

    2007-01-01

    Aromatase (CYP450(arom), CYP19) is an enzyme responsible for converting the aliphatic androgens androstenedione and testosterone to the aromatic estrogens estrone and estradiol, respectively. These endogenous hormones are a key factor in cancer tumor formation and proliferation through a cascade starting from estrogen binding to estrogen receptor. To interfere with the overproduction of estrogens especially in tumor tissue, it is possible to inhibit aromatase activity. This can be achieved using aromatase inhibitors. In order to design novel aromatase inhibitors, it is necessary to have an understanding of the active site of aromatase. As no crystal structure of the enzyme has yet been published, we built a homology model of aromatase using the first crystallized mammalian cytochrome enzyme, rabbit 21-progesterone hydroxylase 2C5, as a template structure. The initial model was validated with exhaustive molecular dynamics simulation with and without the natural substrate androstenedione. The resulting enzyme-substrate complex shows very good stability and only two of the residues are in disallowed regions in a Ramachandran plot.

  19. Understanding the pathological manifestations of aromatase excess syndrome: lessons for clinical diagnosis

    PubMed Central

    Shozu, Makio; Fukami, Maki; Ogata, Tsutomu

    2014-01-01

    CYP19A1 Aromatase excess syndrome is characterized by pre- or peripubertal onset of gynecomastia due to estrogen excess because of a gain-of-function mutation in the aromatase gene (CYP19A1). Subchromosomal recombination events including duplication, deletion, and inversion has been identified. The latter two recombinations recruit novel promoters for CYP19A1 through a unique mechanism. Gynecomastia continues for life, and although the general condition is well preserved, it may cause psychological issues. Minor symptoms (variably advanced bone age and short adult height), if present, are exclusively because of estrogen excess. Serum estradiol levels are elevated in 48% of affected males, but are not necessarily useful for diagnosis. Molecular analysis of CYP19A1 mutations is mandatory to confirm aromatase excess syndrome diagnosis. Furthermore, the use of an aromatase inhibitor can ameliorate gynecomastia. PMID:25264451

  20. The binding of lignans, flavonoids and coumestrol to CYP450 aromatase: a molecular modelling study.

    PubMed

    Karkola, Sampo; Wähälä, Kristiina

    2009-03-25

    Androgens are transformed into aromatic estrogens by CYP450 aromatase in a three-step reaction consuming three equivalents of oxygen and three equivalents of NADPH. Estrogens are substrates for nuclear estrogen receptors (ERs) and play a key role in estrogen-dependent tumour cell formation and proliferation. Natural phytoestrogens are proved to be competitive inhibitors of aromatase enzyme at IC(50) values in micromolar levels. In order to understand the mechanisms involved in the binding of various phytoestrogens, we used our model of CYP450 aromatase to study the binding of phytoestrogens using molecular dynamics simulations with a bound phytoestrogen. The simulation trajectory was analysed to find the essential interactions which take place upon binding and a representative structure of the trajectory was minimized for docking studies. Sets of phytoestrogens, such as lignans, flavonoids/isoflavonoids and coumestrol, were docked into the aromatase active site and the binding modes were studied.

  1. Characterization of aromatase binding agents from the dichloromethane extract of Corydalis yanhusuo using ultrafiltration and liquid chromatography tandem mass spectrometry.

    PubMed

    Shi, Jing; Zhang, Xiaoyu; Ma, Zhongjun; Zhang, Min; Sun, Fang

    2010-05-14

    Aromatase represents an important target for the treatment of hormone-dependent breast cancer. In the present study, nine alkaloids from the dichloromethane extract of Corydalis yanhusuo were identified by liquid chromatography tandem mass spectrometry (LC-MS/MS) and tested for their aromatase binding activities using an ultrafiltration LC-MS method by investigating the differences of peak areas of compounds before and after incubations with aromatase. It was demonstrated that the quaternary protoberberine alkaloids and the tertiary protoberberine alkaloids exhibited potent aromatase binding activities. The quaternary ammonium group and the methyl group at C-13 position of tertiary protoberberine alkaloids might be necessary for the activity. The findings should provide guidance for the discovery of potential aromatase inhibitors from natural products.

  2. Structural basis for androgen specificity and oestrogen synthesis in human aromatase

    SciTech Connect

    Ghosh, Debashis; Griswold, Jennifer; Erman, Mary; Pangborn, Walter

    2009-03-06

    Aromatase cytochrome P450 is the only enzyme in vertebrates known to catalyse the biosynthesis of all oestrogens from androgens. Aromatase inhibitors therefore constitute a frontline therapy for oestrogen-dependent breast cancer. In a three-step process, each step requiring 1 mol of O{sub 2}, 1 mol of NADPH, and coupling with its redox partner cytochrome P450 reductase, aromatase converts androstenedione, testosterone and 16{alpha}-hydroxytestosterone to oestrone, 17{beta}-oestradiol and 17{beta},16{alpha}-oestriol, respectively. The first two steps are C19-methyl hydroxylation steps, and the third involves the aromatization of the steroid A-ring, unique to aromatase. Whereas most P450s are not highly substrate selective, it is the hallmark androgenic specificity that sets aromatase apart. The structure of this enzyme of the endoplasmic reticulum membrane has remained unknown for decades, hindering elucidation of the biochemical mechanism. Here we present the crystal structure of human placental aromatase, the only natural mammalian, full-length P450 and P450 in hormone biosynthetic pathways to be crystallized so far. Unlike the active sites of many microsomal P450s that metabolize drugs and xenobiotics, aromatase has an androgen-specific cleft that binds the androstenedione molecule snugly. Hydrophobic and polar residues exquisitely complement the steroid backbone. The locations of catalytically important residues shed light on the reaction mechanism. The relative juxtaposition of the hydrophobic amino-terminal region and the opening to the catalytic cleft shows why membrane anchoring is necessary for the lipophilic substrates to gain access to the active site. The molecular basis for the enzyme's androgenic specificity and unique catalytic mechanism can be used for developing next-generation aromatase inhibitors.

  3. Structural basis for androgen specificity and oestrogen synthesis in human aromatase.

    PubMed

    Ghosh, Debashis; Griswold, Jennifer; Erman, Mary; Pangborn, Walter

    2009-01-08

    Aromatase cytochrome P450 is the only enzyme in vertebrates known to catalyse the biosynthesis of all oestrogens from androgens. Aromatase inhibitors therefore constitute a frontline therapy for oestrogen-dependent breast cancer. In a three-step process, each step requiring 1 mol of O(2), 1 mol of NADPH, and coupling with its redox partner cytochrome P450 reductase, aromatase converts androstenedione, testosterone and 16alpha-hydroxytestosterone to oestrone, 17beta-oestradiol and 17beta,16alpha-oestriol, respectively. The first two steps are C19-methyl hydroxylation steps, and the third involves the aromatization of the steroid A-ring, unique to aromatase. Whereas most P450s are not highly substrate selective, it is the hallmark androgenic specificity that sets aromatase apart. The structure of this enzyme of the endoplasmic reticulum membrane has remained unknown for decades, hindering elucidation of the biochemical mechanism. Here we present the crystal structure of human placental aromatase, the only natural mammalian, full-length P450 and P450 in hormone biosynthetic pathways to be crystallized so far. Unlike the active sites of many microsomal P450s that metabolize drugs and xenobiotics, aromatase has an androgen-specific cleft that binds the androstenedione molecule snugly. Hydrophobic and polar residues exquisitely complement the steroid backbone. The locations of catalytically important residues shed light on the reaction mechanism. The relative juxtaposition of the hydrophobic amino-terminal region and the opening to the catalytic cleft shows why membrane anchoring is necessary for the lipophilic substrates to gain access to the active site. The molecular basis for the enzyme's androgenic specificity and unique catalytic mechanism can be used for developing next-generation aromatase inhibitors.

  4. Possible role of the aromatase-independent steroid metabolism pathways in hormone responsive primary breast cancers.

    PubMed

    Hanamura, Toru; Niwa, Toshifumi; Gohno, Tatsuyuki; Kurosumi, Masafumi; Takei, Hiroyuki; Yamaguchi, Yuri; Ito, Ken-ichi; Hayashi, Shin-ichi

    2014-01-01

    Aromatase inhibitors (AIs) exert antiproliferative effects by reducing local estrogen production from androgens in postmenopausal women with hormone-responsive breast cancer. Previous reports have shown that androgen metabolites generated by the aromatase-independent enzymes, 5α-androstane-3β, 17β-diol (3β-diol), androst-5-ene-3β, and 17β-diol (A-diol), also activate estrogen receptor (ER) α. Estradiol (E2) can also reportedly be generated from estrone sulfate (E1S) pooled in the plasma. Estrogenic steroid-producing aromatase-independent pathways have thus been proposed as a mechanism of AI resistance. However, it is unclear whether these pathways are functional in clinical breast cancer. To investigate this issue, we assessed the transcriptional activities of ER in 45 ER-positive human breast cancers using the adenovirus estrogen-response element-green fluorescent protein assay and mRNA expression levels of the ER target gene, progesterone receptor, as indicators of ex vivo and in vivo ER activity, respectively. We also determined mRNA expression levels of 5α-reductase type 1 (SRD5A1) and 3β-hydroxysteroid dehydrogenase type 1 (3β-HSD type 1; HSD3B1), which produce 3β-diol from androgens, and of steroid sulfatase (STS) and 17β-hydroxysteroid dehydrogenase type 1 (17β-HSD type 1; HSD17B1), which produce E2 or A-diol from E1S or dehydroepiandrosterone sulfate. SRD5A1 and HSD3B1 expression levels were positively correlated with ex vivo and in vivo ER activities. STS and HSD17B1 expression levels were positively correlated with in vivo ER activity alone. Elevated expression levels of these steroid-metabolizing enzymes in association with high in vivo ER activity were particularly notable in postmenopausal patients. Analysis of the expression levels of steroid-metabolizing enzymes revealed positive correlations between SRD5A1 and HSD3B1, and STS and HSD17B1. These findings suggest that the SRD5A1-HSD3B1 as well as the STS-HSD17B pathways, could contributes

  5. Brain and gonadal aromatase as potential targets of endocrine disrupting chemicals in a model species, the zebrafish (Danio rerio).

    PubMed

    Hinfray, N; Palluel, O; Turies, C; Cousin, C; Porcher, J M; Brion, F

    2006-08-01

    Many chemicals in the aquatic environment are able to adversely affect in vitro brain and ovarian aromatase expression/activity. However, it remains to be determined if these substances elicit in vivo effect in fish. With the view to further understanding possible effects of endocrine disrupting chemicals (EDCs) on aromatase function, we first developed methods to measure brain and ovarian aromatase expression/activity in a model species, the zebrafish, and assessed the effect of estradiol (E2) and androstatrienedione (ATD), a steroidal aromatase inhibitor. We showed that CYP19b gene was predominantly expressed in the brain whereas in the ovary CYP19a mRNA level was predominant. Moreover, aromatase activities (AA) were higher in brain than in ovary. In adult zebrafish, E2 treatment had no effect on aromatase expression/activity in brain, whereas at larval stage, E2 strongly triggered CYP19b expression. In the ovaries, E2 led to a complete inhibition of both CYP19a expression and AA. Exposure to ATD led to a total inhibition of both brain and ovarian AA but had no effect on CYP19 transcripts abundance. Together, these results provide relevant knowledge concerning the characterization of aromatase in the zebrafish, and reinforce the idea that brain and ovarian aromatase are promising markers of EDCs in fish and deserve further in vivo studies.

  6. Kinesin-5 inhibitor resistance is driven by kinesin-12

    PubMed Central

    Sturgill, Emma G.; Norris, Stephen R.; Guo, Yan

    2016-01-01

    The microtubule (MT) cytoskeleton bipolarizes at the onset of mitosis to form the spindle. In animal cells, the kinesin-5 Eg5 primarily drives this reorganization by actively sliding MTs apart. Its primacy during spindle assembly renders Eg5 essential for mitotic progression, demonstrated by the lethal effects of kinesin-5/Eg5 inhibitors (K5Is) administered in cell culture. However, cultured cells can acquire resistance to K5Is, indicative of alternative spindle assembly mechanisms and/or pharmacological failure. Through characterization of novel K5I-resistant cell lines, we unveil an Eg5 motility-independent spindle assembly pathway that involves both an Eg5 rigor mutant and the kinesin-12 Kif15. This pathway centers on spindle MT bundling instead of Kif15 overexpression, distinguishing it from those previously described. We further show that large populations (∼107 cells) of HeLa cells require Kif15 to survive K5I treatment. Overall, this study provides insight into the functional plasticity of mitotic kinesins during spindle assembly and has important implications for the development of antimitotic regimens that target this process. PMID:27091450

  7. Theoretical Study of the Mechanism of Exemestane Hydroxylation Catalyzed by Human Aromatase Enzyme.

    PubMed

    Viciano, Ignacio; Martí, Sergio

    2016-04-07

    Human aromatase (CYP19A1) aromatizes the androgens to form estrogens via a three-step oxidative process. The estrogens are necessary in humans, mainly in women, because of the role they play in sexual and reproductive development. However, these also are involved in the development and growth of hormone-dependent breast cancer. Therefore, inhibition of the enzyme aromatase, by means of drugs known as aromatase inhibitors, is the frontline therapy for these types of cancers. Exemestane is a suicidal third-generation inhibitor of aromatase, currently used in breast cancer treatment. In this study, the hydroxylation of exemestane catalyzed by aromatase has been studied by means of hybrid QM/MM methods. The Free Energy Perturbation calculations provided a free energy of activation for the hydrogen abstraction step (rate-limiting step) of 17 kcal/mol. The results reveal that the hydroxylation of exemestane is not the inhibition stage, suggesting a possible competitive mechanism between the inhibitor and the natural substrate androstenedione in the first catalytic subcycle of the enzyme. Furthermore, the analysis of the interaction energy for the substrate and the cofactor in the active site shows that the role of the enzymatic environment during this reaction consists of a transition state stabilization by means of electrostatic effects.

  8. Higher order organization of human placental aromatase.

    PubMed

    Ghosh, Debashis; Jiang, Wenhua; Lo, Jessica; Egbuta, Chinaza

    2011-07-01

    Aromatase (CYP19A1) is an integral membrane enzyme that catalyzes the removal of the 19-methyl group and aromatization of the A-ring of androgens. All human estrogens are synthesized from their androgenic precursors by this unique cytochrome P450. The crystal structure of active aromatase purified from human placenta has recently been determined in complex with its natural substrate androstenedione in the high-spin ferric state of heme. Hydrogen bond forming interactions and tight packing hydrophobic side chains closely complement puckering of the steroid backbone, thereby providing the molecular basis for the androgenic specificity of aromatase. In the crystal, aromatase molecules are linked by a head-to-tail intermolecular interaction via a surface loop between helix D and helix E of one aromatase molecule that penetrates the heme-proximal cavity of the neighboring, crystallographically related molecule, thus forming in tandem a polymeric aromatase chain. This intermolecular interaction is similar to the aromatase-cytochrome P450 reductase coupling and is driven by electrostatics between the negative potential surface of the D-E loop region and the positively charged heme-proximal cavity. This loop-to-proximal site link in aromatase is rather unique--there are only a few of examples of somewhat similar intermolecular interactions in the entire P450 structure database. Furthermore, the amino acids involved in the intermolecular contact appear to be specific for aromatase. Higher order organization of aromatase monomers may have implications in lipid integration and catalysis.

  9. Bacterial self-resistance to the natural proteasome inhibitor salinosporamide A

    PubMed Central

    Kale, Andrew J.; McGlinchey, Ryan P.; Lechner, Anna; Moore, Bradley S.

    2011-01-01

    Proteasome inhibitors have recently emerged as a therapeutic strategy in cancer chemotherapy but susceptibility to drug resistance limits their efficacy. The marine actinobacterium Salinispora tropica produces salinosporamide A (NPI-0052, marizomib), a potent proteasome inhibitor and promising clinical agent in the treatment of multiple myeloma. Actinobacteria also possess 20S proteasome machinery, raising the question of self-resistance. We identified a redundant proteasome β-subunit, SalI, encoded within the salinosporamide biosynthetic gene cluster and biochemically characterized the SalI proteasome complex. The SalI β-subunit has an altered substrate specificity profile, 30-fold resistance to salinosporamide A, and cross-resistance to the FDA-approved proteasome inhibitor bortezomib. An A49V mutation in SalI correlates to clinical bortezomib resistance from a human proteasome β 5-subunit A49T mutation, suggesting that intrinsic resistance to natural proteasome inhibitors may predict clinical outcomes. PMID:21882868

  10. Resistance of human glioblastoma multiforme cells to growth factor inhibitors is overcome by blockade of inhibitor of apoptosis proteins

    PubMed Central

    Ziegler, David S.; Wright, Renee D.; Kesari, Santosh; Lemieux, Madeleine E.; Tran, Mary A.; Jain, Monish; Zawel, Leigh; Kung, Andrew L.

    2008-01-01

    Multiple receptor tyrosine kinases (RTKs), including PDGFR, have been validated as therapeutic targets in glioblastoma multiforme (GBM), yet inhibitors of RTKs have had limited clinical success. As various antiapoptotic mechanisms render GBM cells resistant to chemo- and radiotherapy, we hypothesized that these antiapoptotic mechanisms also confer resistance to RTK inhibition. We found that in vitro inhibition of PDGFR in human GBM cells initiated the intrinsic pathway of apoptosis, as evidenced by mitochondrial outer membrane permeabilization, but downstream caspase activation was blocked by inhibitor of apoptosis proteins (IAPs). Consistent with this, inhibition of PDGFR combined with small molecule inactivation of IAPs induced apoptosis in human GBM cells in vitro and had synergistic antitumor effects in orthotopic mouse models of GBM and in primary human GBM neurospheres. These results demonstrate that concomitant inhibition of IAPs can overcome resistance to RTK inhibitors in human malignant GBM cells, and suggest that blockade of IAPs has the potential to improve treatment outcomes in patients with GBM. PMID:18677408

  11. Pharmacophore mapping of flavone derivatives for aromatase inhibition.

    PubMed

    Nagar, Shuchi; Islam, Md Ataul; Das, Suvadra; Mukherjee, Arup; Saha, Achintya

    2008-02-01

    Aromatase, which catalyses the final step in the steroidogenesis pathway of estrogen, has been target for the design of inhibitor in the treatment of hormone dependent breast cancer for postmenopausal women. The extensive SAR studies performed in the last 30 years to search for potent, selective and less toxic compounds, have led to the development of second and third generation of non-steroidal aromatase inhibitors (AI). Besides the development of synthetic compounds, several naturally occurring and synthetic flavonoids, which are ubiquitous natural phenolic compounds and mediate the host of biological activities, are found to demonstrate inhibitory effects on aromatase. The present study explores the pharmacophores, i.e., the structural requirements of flavones (Fig. 1) for inhibition of aromatase activity, using quantitative structure activity relationship (QSAR) and space modeling approaches. The classical QSAR studies generate the model (R (2) = 0.924, Q (2) = 0.895, s = 0.233) that shows the importance of aromatic rings A and C, along with substitutional requirements in meta and para positions of ring C for the activity. 3D QSAR of Comparative Molecular Field Analysis (CoMFA, R (2) = 0.996, R(2)(cv) = 0.791) and Comparative Molecular Similarity Analysis (CoMSIA, R (2) = 0.992, R(2)(cv) = 0.806) studies show contour maps of steric and hydrophobic properties and contribution of acceptor and donor of the molecule, suggesting the presence of steric hindrance due to ring C and R''-substituent, bulky hydrophobic substitution in ring A, along with acceptors at positions 11, and alpha and gamma of imidazole ring, and donor in ring C favor the inhibitory activity. Further space modeling (CATALYST) study (R = 0.941, Delta( cost ) = 96.96, rmsd = 0.876) adjudge the presence of hydrogen bond acceptor (keto functional group), hydrophobic (ring A) and aromatic rings (steric hindrance) along with critical distance among features are important for the inhibitory activity.

  12. Mechanisms of Epidermal Growth Factor Receptor Tyrosine Kinase Inhibitor Resistance and Strategies to Overcome Resistance in Lung Adenocarcinoma

    PubMed Central

    Chang, Yoon Soo; Choi, Chang-Min

    2016-01-01

    Somatic mutations that lead to hyperactivation of epidermal growth factor receptor (EGFR) signaling are detected in approximately 50% of lung adenocarcinoma in people from the Far East population and tyrosine kinase inhibitors are now the standard first line treatment for advanced disease. They have led to a doubling of progression-free survival and an increase in overall survival by more than 2 years. However, emergence of resistant clones has become the primary cause for treatment failure, and has created a new challenge in the daily management of patients with EGFR mutations. Identification of mechanisms leading to inhibitor resistance has led to new therapeutic modalities, some of which have now been adapted for patients with unsuccessful tyrosine kinase inhibitor treatment. In this review, we describe mechanisms of tyrosine kinase inhibitor resistance and the available strategies to overcoming resistance. PMID:27790276

  13. A novel method for measuring aromatase activity in tissue samples by determining estradiol concentrations.

    PubMed

    Tinwell, H; Rascle, J B; Colombel, S; Al Khansa, I; Freyberger, A; Bars, R

    2011-07-01

    Increasing scrutiny of endocrine disrupters has led to changes to European pesticide and biocide legislation and to the introduction of the Endocrine Disrupter Screening Program by the US EPA. One element of endocrine disrupter identification is to determine its effects on aromatase, but most available assays are limited as they depend on tritiated water production to indicate enzyme activity. Whilst acceptable for determining aromatase effects using a cell-free approach, this method is unreliable for cell or tissue-based investigations as other cytochrome P-450 isoenzyme activities can similarly produce tritiated water and consequently confound interpretation of the aromatase data. To address this lack of specificity an assay directly measuring the final estrogen product by incubating rat tissue protein with testosterone and measuring the resultant estradiol concentration was developed. Using this approach we demonstrated marked increases in enzyme activity in pregnant rat ovary samples and dose-related inhibitions when incubating non-pregnant rat ovary samples with known aromatase inhibitors. Hepatic aromatase activity was investigated using our method and by tritiated water production with microsomes from rats dosed with the antiandrogen 1,1-dichloro-2,2-bis(4 chlorophenyl)ethane. Additional cytochrome P-450s were also measured. Treatment-related increased tritiated water production and general hepatic enzyme activity were recorded but estradiol was not increased, indicating that the increased tritiated water was due to general enzyme activity and not aromatase activity. A simple and specific method has been developed that can detect aromatase inhibition and induction, which when applied to tissue samples, provides a means of generating relevant animal data concerning chemical effects on the aromatase enzyme.

  14. SIRT1 positively regulates breast cancer associated human aromatase (CYP19A1) expression.

    PubMed

    Holloway, Kimberly R; Barbieri, Andreia; Malyarchuk, Svitlana; Saxena, Madhurima; Nedeljkovic-Kurepa, Ana; Cameron Mehl, Mathieu; Wang, Allison; Gu, Xin; Pruitt, Kevin

    2013-03-01

    Breast cancer remains one of the leading causes of death in women diagnosed with cancer. In breast cancer, aberrant expression of the CYP19A1 gene, which encodes the aromatase enzyme, contributes to increased intratumoral levels of estradiol. Regardless of whether this estrogen is produced by peripheral tissues or within specific subpopulations of cells within the breast tumor, it is clear that the aromatase enzymatic activity is critical for the growth of estrogen-dependent tumors. Currently, aromatase inhibitors have proven to be highly effective in blocking the growth of estrogen-dependent forms of breast cancer. CYP19A1 transcription is tightly controlled by 10 tissue-specific promoters. In breast cancer, however, aromatase transcription is driven by multiple promoters that somehow override the tissue-specific regulation of normal tissue. Here, we explore the role that the deacetylase, sirtuin-1 (SIRT1), plays in positively regulating aromatase in breast cancer. We demonstrate that the use of cambinol and the SIRT1/2 inhibitor VII, 2 small molecule inhibitors of SIRT1 and SIRT2, as well as small molecule inhibitors and small interfering RNA specific to SIRT1, all reduce the levels of aromatase mRNA. We further demonstrate that pharmacologic inhibition causes a marked reduction in aromatase protein levels. Additionally, by chromatin immunoprecipitation, we demonstrate that SIRT1 occupies the promoter regions PI.3/PII and PI.4, and its inhibition leads to increased acetylation of estrogen-related receptorα, a transcription factor that positively regulates CYP19A1 transcription in epithelial cells. Finally, we demonstrate by immunohistochemistry that SIRT1 is significantly up-regulated in invasive ductal carcinoma relative to normal tissue adjacent to tumor, further suggesting a role of SIRT1 in breast cancer. This work uncovers a new mechanism for the regulation of aromatase and provides rationale for further investigation of how the inhibition of specific

  15. Adaptive and Acquired Resistance to EGFR Inhibitors Converge on the MAPK Pathway

    PubMed Central

    Ma, Pengfei; Fu, Yujie; Chen, Minjiang; Jing, Ying; Wu, Jie; Li, Ke; Shen, Ying; Gao, Jian-Xin; Wang, Mengzhao; Zhao, Xiaojing; Zhuang, Guanglei

    2016-01-01

    Both adaptive and acquired resistance significantly limits the efficacy of the epidermal growth factor receptor (EGFR) kinase inhibitors. However, the distinct or common mechanisms of adaptive and acquired resistance have not been fully characterized. Here, through systematic modeling of erlotinib resistance in lung cancer, we found that feedback reactivation of MAPK signaling following erlotinib treatment, which was dependent on the MET receptor, contributed to the adaptive resistance of EGFR inhibitors. Interestingly, acquired resistance to erlotinib was also associated with the MAPK pathway activation as a result of CRAF or NRAS amplification. Consequently, combined inhibition of EGFR and MAPK impeded the development of both adaptive and acquired resistance. These observations demonstrate that adaptive and acquired resistance to EGFR inhibitors can converge on the same pathway and credential cotargeting EGFR and MAPK as a promising therapeutic approach in EGFR mutant tumors. PMID:27279914

  16. Clotrimazole exposure modulates aromatase activity in gonads and brain during gonadal differentiation in Xenopus tropicalis frogs.

    PubMed

    Gyllenhammar, Irina; Eriksson, Hanna; Söderqvist, Anneli; Lindberg, Richard H; Fick, Jerker; Berg, Cecilia

    2009-01-31

    Clotrimazole is a pharmaceutical used for treatment of fungal infections. It has been found in surface waters outside municipal wastewater treatment plants but data are scarce regarding its effects on aquatic organisms. It is known that clotrimazole and other imidazole fungicides are inhibitors of the enzyme aromatase (CYP 19). Aromatase converts androgens into estrogens and is suggested to be involved in the sex differentiation in amphibians. The aim of the present study was to evaluate effects of larval exposure to clotrimazole on aromatase activity in brain and gonads, and on gonadal differentiation in Xenopus tropicalis frogs. Another purpose was to determine if larval exposure to ethynylestradiol (EE(2)), at a concentration known to cause male-to-female sex reversal, affects aromatase activity in brain and gonads during gonadal differentiation. Tadpoles were exposed from shortly after hatching (Nieuwkoop and Faber developmental stages 47-48) until complete metamorphosis (NF stage 66) to 6, 41, and 375 nM clotrimazole or 100 nM (nominal) EE(2). Aromatase activity was measured in the brain and gonad/kidney complex of tadpoles during gonadal differentiation (NF stage 56) and, in the clotrimazole experiment, also at metamorphosis. In clotrimazole-exposed tadpoles gonadal aromatase activity increased over exposure time in the 41 and 375 nM groups but did not differ significantly from the control group. Gonadal aromatase activity was increased in both sexes exposed to 41 and 375 nM clotrimazole at metamorphosis. Brain aromatase activity was decreased in tadpoles (NF stage 56) exposed to 375 nM clotrimazole, but at metamorphosis no differences were seen between groups or between sexes. No effects of clotrimazole on sex ratio or gonadal histology were noted at completed metamorphosis. EE(2)-exposed tadpoles had a slightly decreased gonadal aromatase activity, though not significantly different from control group, and there was no effect of EE(2) on brain aromatase

  17. Sulfonamide-Based Inhibitors of Aminoglycoside Acetyltransferase Eis Abolish Resistance to Kanamycin in Mycobacterium tuberculosis.

    PubMed

    Garzan, Atefeh; Willby, Melisa J; Green, Keith D; Gajadeera, Chathurada S; Hou, Caixia; Tsodikov, Oleg V; Posey, James E; Garneau-Tsodikova, Sylvie

    2016-12-08

    A two-drug combination therapy where one drug targets an offending cell and the other targets a resistance mechanism to the first drug is a time-tested, yet underexploited approach to combat or prevent drug resistance. By high-throughput screening, we identified a sulfonamide scaffold that served as a pharmacophore to generate inhibitors of Mycobacterium tuberculosis acetyltransferase Eis, whose upregulation causes resistance to the aminoglycoside (AG) antibiotic kanamycin A (KAN) in Mycobacterium tuberculosis. Rational systematic derivatization of this scaffold to maximize Eis inhibition and abolish the Eis-mediated KAN resistance of M. tuberculosis yielded several highly potent agents. A crystal structure of Eis in complex with one of the most potent inhibitors revealed that the inhibitor bound Eis in the AG-binding pocket held by a conformationally malleable region of Eis (residues 28-37) bearing key hydrophobic residues. These Eis inhibitors are promising leads for preclinical development of innovative AG combination therapies against resistant TB.

  18. Anhedonia Predicts Poorer Recovery among Youth with Selective Serotonin Reuptake Inhibitor Treatment-Resistant Depression

    ERIC Educational Resources Information Center

    McMakin, Dana L.; Olino, Thomas M.; Porta, Giovanna; Dietz, Laura J.; Emslie, Graham; Clarke, Gregory; Wagner, Karen Dineen; Asarnow, Joan R.; Ryan, Neal D.; Birmaher, Boris; Shamseddeen, Wael; Mayes, Taryn; Kennard, Betsy; Spirito, Anthony; Keller, Martin; Lynch, Frances L.; Dickerson, John F.; Brent, David A.

    2012-01-01

    Objective: To identify symptom dimensions of depression that predict recovery among selective serotonin reuptake inhibitor (SSRI) treatment-resistant adolescents undergoing second-step treatment. Method: The Treatment of Resistant Depression in Adolescents (TORDIA) trial included 334 SSRI treatment-resistant youth randomized to a medication…

  19. Targeting SHP2 for EGFR inhibitor resistant non-small cell lung carcinoma

    SciTech Connect

    Xu, Jie; Zeng, Li-Fan; Shen, Weihua; Turchi, John J.; Zhang, Zhong-Yin

    2013-10-04

    Highlights: •SHP2 is required for EGFR inhibitor resistant NSCLC H1975 cell proliferation. •SHP2 inhibitor blocks EGF-stimulated ERK1/2 activation and proliferation. •SHP2 inhibitor exhibits marked anti-tumor activity in H1975 xenograft mice. •SHP2 inhibitor synergizes with PI3K inhibitor in suppressing cell growth. •Targeting SHP2 represents a novel strategy for EGFR inhibitor resistant NSCLCs. -- Abstract: Targeted therapy with inhibitors of epidermal growth factor receptor (EGFR) has produced a noticeable benefit to non-small cell lung cancer (NSCLC) patients whose tumors carry activating mutations (e.g. L858R) in EGFR. Unfortunately, these patients develop drug resistance after treatment, due to acquired secondary gatekeeper mutations in EGFR (e.g. T790M). Given the critical role of SHP2 in growth factor receptor signaling, we sought to determine whether targeting SHP2 could have therapeutic value for EGFR inhibitor resistant NSCLC. We show that SHP2 is required for EGF-stimulated ERK1/2 phosphorylation and proliferation in EGFR inhibitor resistant NSCLC cell line H1975, which harbors the EGFR T790M/L858R double-mutant. We demonstrate that treatment of H1975 cells with II-B08, a specific SHP2 inhibitor, phenocopies the observed growth inhibition and reduced ERK1/2 activation seen in cells treated with SHP2 siRNA. Importantly, we also find that II-B08 exhibits marked anti-tumor activity in H1975 xenograft mice. Finally, we observe that combined inhibition of SHP2 and PI3K impairs both the ERK1/2 and PI3K/AKT signaling axes and produces significantly greater effects on repressing H1975 cell growth than inhibition of either protein individually. Collectively, these results suggest that targeting SHP2 may represent an effective strategy for treatment of EGFR inhibitor resistant NSCLCs.

  20. Characterization of a mantle cell lymphoma cell line resistant to the Chk1 inhibitor PF-00477736.

    PubMed

    Restelli, Valentina; Chilà, Rosaria; Lupi, Monica; Rinaldi, Andrea; Kwee, Ivo; Bertoni, Francesco; Damia, Giovanna; Carrassa, Laura

    2015-11-10

    Mantle cell lymphoma (MCL) is an aggressive B-cell lymphoma characterized by the chromosomal translocation t(11;14) that leads to constitutive expression of cyclin D1, a master regulator of the G1-S phase. Chk1 inhibitors have been recently shown to be strongly effective as single agents in MCL. To investigate molecular mechanisms at the basis of Chk1 inhibitor activity, a MCL cell line resistant to the Chk1 inhibitor PF-00477736 (JEKO-1 R) was obtained and characterized. The JEKO-1 R cell line was cross resistant to another Chk1 inhibitor (AZD-7762) and to the Wee1 inhibitor MK-1775. It displayed a shorter doubling time than parental cell line, likely due to a faster S phase. Cyclin D1 expression levels were decreased in resistant cell line and its re-overexpression partially re-established PF-00477736 sensitivity. Gene expression profiling showed an enrichment in gene sets involved in pro-survival pathways in JEKO-1 R. Dasatinib treatment partly restored PF-00477736 sensitivity in resistant cells suggesting that the pharmacological interference of pro-survival pathways can overcome the resistance to Chk1 inhibitors. These data further corroborate the involvement of the t(11;14) in cellular sensitivity to Chk1 inhibitors, fostering the clinical testing of Chk1 inhibitors as single agents in MCL.

  1. Hepatitis C virus NS5A inhibitors and drug resistance mutations.

    PubMed

    Nakamoto, Shingo; Kanda, Tatsuo; Wu, Shuang; Shirasawa, Hiroshi; Yokosuka, Osamu

    2014-03-21

    Some direct-acting antiviral agents for hepatitis C virus (HCV), such as telaprevir and boceprevir have been available since 2011. It was reported that HCV NS5A is associated with interferon signaling related to HCV replication and hepatocarcinogenesis. HCV NS5A inhibitors efficiently inhibited HCV replication in vitro. Human studies showed that dual, triple and quad regimens with HCV NS5A inhibitors, such as daclatasvir and ledipasvir, in combination with other direct-acting antiviral agents against other regions of HCV with or without peginterferon/ribavirin, could efficiently inhibit HCV replication according to HCV genotypes. These combinations might be a powerful tool for "difficult-to-treat" HCV-infected patients. "First generation" HCV NS5A inhibitors such as daclatasvir, ledipasvir and ABT-267, which are now in phase III clinical trials, could result in resistance mutations. "Second generation" NS5A inhibitors such as GS-5816, ACH-3102, and MK-8742, have displayed improvements in the genetic barrier while maintaining potency. HCV NS5A inhibitors are safe at low concentrations, which make them attractive for use despite low genetic barriers, although, in fact, HCV NS5A inhibitors should be used with HCV NS3/4A inhibitors, HCV NS5B inhibitors or peginterferon plus ribavirin. This review article describes HCV NS5A inhibitor resistance mutations and recommends that HCV NS5A inhibitors be used in combination regimens potent enough to prevent the emergence of resistant variants.

  2. Peripubertal aromatase inhibition in male rats has adverse long-term effects on bone strength and growth and induces prostatic hyperplasia.

    PubMed

    Bajpai, Anurag; Simm, Peter J; McPherson, Stephen J; Russo, Vincenzo C; Azar, Walid J; Wark, John D; Risbridger, Gail P; Werther, George A

    2010-10-01

    Aromatase inhibitors have been increasingly used in boys with growth retardation to prolong the duration of growth and increase final height. Multiple important roles of oestrogen in males point to potential adverse effects of this strategy. Although the deleterious effects of aromatase deficiency in early childhood and adulthood are well documented, there is limited information about the potential long-term adverse effects of peripubertal aromatase inhibition. To address this issue, we evaluated short-term and long-term effects of peripubertal aromatase inhibition in an animal model. Peripubertal male Wistar rats were treated with aromatase inhibitor letrozole or placebo and followed until adulthood. Letrozole treatment caused sustained reduction in bone strength and alteration in skeletal geometry, lowering of IGF1 levels, inhibition of growth resulting in significantly lower weight and length of treated animals and development of focal prostatic hyperplasia. Our observation of adverse long-term effects after peripubertal male rats were exposed to aromatase inhibitors highlights the need for further characterisation of long-term adverse effects of aromatase inhibitors in peripubertal boys before further widespread use is accepted. Furthermore, this suggests the need to develop more selective oestrogen inhibition strategies in order to inhibit oestrogen action on the growth plate, while beneficial effects in other tissues are preserved.

  3. Activation of Pim Kinases Is Sufficient to Promote Resistance to MET Small Molecule Inhibitors

    PubMed Central

    An, Ningfei; Xiong, Ying; LaRue, Amanda C.; Kraft, Andrew S.; Cen, Bo

    2015-01-01

    MET blockade offers a new targeted therapy particularly in those cancers with MET amplification. However, the efficacy and the duration of the response to MET inhibitors are limited by the emergence of drug resistance. Here we report that resistance to small molecule inhibitors of MET can arise from increased expression of the pro-survival Pim protein kinases. This resistance mechanism was documented in non-small cell lung cancer and gastric cancer cells with MET amplification. Inhibition of Pim kinases enhanced cell death triggered by short-term treatment with MET inhibitors. Pim kinases control the translation of anti-apoptotic protein Bcl-2 at an internal ribosome entry site and this mechanism was identified as the basis for Pim-mediated resistance to MET inhibitors. Protein synthesis was increased in drug-resistant cells, secondary to a Pim-mediated increase in cap-independent translation. In cells rendered drug resistant by chronic treatment with MET inhibitors, genetic or pharmacological inhibition of Pim kinases was sufficient to restore sensitivity in vitro and in vivo. Taken together, our results rationalize Pim inhibition as a strategy to augment responses and blunt acquired resistance to MET inhibitors in cancer. PMID:26670562

  4. Lenvatinib in combination with golvatinib overcomes hepatocyte growth factor pathway-induced resistance to vascular endothelial growth factor receptor inhibitor.

    PubMed

    Nakagawa, Takayuki; Matsushima, Tomohiro; Kawano, Satoshi; Nakazawa, Youya; Kato, Yu; Adachi, Yusuke; Abe, Takanori; Semba, Taro; Yokoi, Akira; Matsui, Junji; Tsuruoka, Akihiko; Funahashi, Yasuhiro

    2014-06-01

    Vascular endothelial growth factor receptor (VEGFR) inhibitors are approved for the treatment of several tumor types; however, some tumors show intrinsic resistance to VEGFR inhibitors, and some patients develop acquired resistance to these inhibitors. Therefore, a strategy to overcome VEGFR inhibitor resistance is urgently required. Recent reports suggest that activation of the hepatocyte growth factor (HGF) pathway through its cognate receptor, Met, contributes to VEGFR inhibitor resistance. Here, we explored the effect of the HGF/Met signaling pathway and its inhibitors on resistance to lenvatinib, a VEGFR inhibitor. In in vitro experiments, addition of VEGF plus HGF enhanced cell growth and tube formation of HUVECs when compared with stimulation by either factor alone. Lenvatinib potently inhibited the growth of HUVECs induced by VEGF alone, but cells induced by VEGF plus HGF showed lenvatinib resistance. This HGF-induced resistance was cancelled when the Met inhibitor, golvatinib, was added with lenvatinib. Conditioned medium from tumor cells producing high amounts of HGF also conferred resistance to inhibition by lenvatinib. In s.c. xenograft models based on various tumor cell lines with high HGF expression, treatment with lenvatinib alone showed weak antitumor effects, but treatment with lenvatinib plus golvatinib showed synergistic antitumor effects, accompanied by decreased tumor vessel density. These results suggest that HGF from tumor cells confers resistance to tumor endothelial cells against VEGFR inhibitors, and that combination therapy using VEGFR inhibitors with Met inhibitors may be effective for overcoming resistance to VEGFR inhibitors. Further evaluation in clinical trials is warranted.

  5. A three-dimensional model of aromatase cytochrome P450.

    PubMed Central

    Graham-Lorence, S.; Amarneh, B.; White, R. E.; Peterson, J. A.; Simpson, E. R.

    1995-01-01

    P450 hemeproteins comprise a large gene superfamily that catalyzes monooxygenase reactions in the presence of a redox partner. Because the mammalian members are, without exception, membrane-bound proteins, they have resisted structure-function analysis by means of X-ray crystallographic methods. Among P450-catalyzed reactions, the aromatase reaction that catalyzes the conversion of C19 steroids to estrogens is one of the most complex and least understood. Thus, to better understand the reaction mechanism, we have constructed a three-dimensional model of P450arom not only to examine the active site and those residues potentially involved in catalysis, but to study other important structural features such as substrate recognition and redox-partner binding, which require examination of the entire molecule (excepting the putative membrane-spanning region). This model of P450arom was built based on a "core structure" identified from the structures of the soluble, bacterial P450s (P450cam, P450terp, and P450BM-P) rather than by molecular replacement, after which the less conserved elements and loops were added in a rational fashion. Minimization and dynamic simulations were used to optimize the model and the reasonableness of the structure was evaluated. From this model we have postulated a membrane-associated hydrophobic region of aliphatic and aromatic residues involved in substrate recognition, a redox-partner binding region that may be unique compared to other P450s, as well as residues involved in active site orientation of substrates and an inhibitor of P450arom, namely vorozole. We also have proposed a scheme for the reaction mechanism in which a "threonine switch" determines whether oxygen insertion into the substrate molecule involves an oxygen radical or a peroxide intermediate. PMID:7549871

  6. Inhibiting EGF receptor or SRC family kinase signaling overcomes BRAF inhibitor resistance in melanoma

    PubMed Central

    Girotti, Maria R; Pedersen, Malin; Sanchez-Laorden, Berta; Viros, Amaya; Turajlic, Samra; Niculescu-Duvaz, Dan; Zambon, Alfonso; Sinclair, John; Hayes, Andrew; Gore, Martin; Lorigan, Paul; Springer, Caroline; Larkin, James; Jorgensen, Claus; Marais, Richard

    2017-01-01

    We generated cell lines resistant to BRAF inhibitors and show that the EGF receptor (EGFR)–SRC family kinase (SFK)–STAT3 signaling pathway was upregulated in these cells. In addition to driving proliferation of resistant cells, this pathway also stimulated invasion and metastasis. EGFR inhibitors cooperated with BRAF inhibitors to block the growth of the resistant cells in vitro and in vivo, and monotherapy with the broad specificity tyrosine kinase inhibitor dasatinib blocked growth and metastasis in vivo. We analyzed tumors from patients with intrinsic or acquired resistance to vemurafenib and observed increased EGFR and SFK activity. Furthermore, dasatinib blocked the growth and metastasis of one of the resistant tumors in immunocompromised mice. Our data shows that BRAF inhibitor-mediated activation of EFGR/SFK/STAT3 signaling can mediate resistance in BRAF mutant melanoma patients. We describe two treatments that appear to overcome this resistance and could deliver therapeutic efficacy in drug-resistant BRAF mutant melanoma patients. PMID:23242808

  7. Resistance mechanism of human immunodeficiency virus type-1 protease to inhibitors: A molecular dynamic approach

    PubMed Central

    Dayer, Mohammad Reza; Dayer, Mohammad Saaid

    2014-01-01

    Human immunodeficiency virus type 1 (HIV-1) protease inhibitors comprise an important class of drugs used in HIV treatments. However, mutations of protease genes accelerated by low fidelity of reverse transcriptase yield drug resistant mutants of reduced affinities for the inhibitors. This problem is considered to be a serious barrier against HIV treatment for the foreseeable future. In this study, molecular dynamic simulation method was used to examine the combinational and additive effects of all known mutations involved in drug resistance against FDA approved inhibitors. Results showed that drug resistant mutations are not randomly distributed along the protease sequence; instead, they are localized on flexible or hot points of the protein chain. Substitution of more hydrophobic residues in flexible points of protease chains tends to increase the folding, lower the flexibility and decrease the active site area of the protease. The reduced affinities of HIV-1 protease for inhibitors seemed to be due to substantial decrease in the size of the active site and flap mobility. A correlation was found between the binding energy of inhibitors and their affinities for each mutant suggesting the distortion of the active site geometry in drug resistance by preventing effective fitting of inhibitors into the enzymes' active site. To overcome the problem of drug resistance of HIV-1 protease, designing inhibitors of variable functional groups and configurations is proposed. PMID:27843989

  8. Structural Mechanism of the Pan-BCR-ABL Inhibitor Ponatinib (AP24534): Lessons for Overcoming Kinase Inhibitor Resistance

    SciTech Connect

    Zhou, Tianjun; Commodore, Lois; Huang, Wei-Sheng; Wang, Yihan; Thomas, Mathew; Keats, Jeff; Xu, Qihong; Rivera, Victor M.; Shakespeare, William C.; Clackson, Tim; Dalgarno, David C.; Zhu, Xiaotian

    2012-01-20

    The BCR-ABL inhibitor imatinib has revolutionized the treatment of chronic myeloid leukemia. However, drug resistance caused by kinase domain mutations has necessitated the development of new mutation-resistant inhibitors, most recently against the T315I gatekeeper residue mutation. Ponatinib (AP24534) inhibits both native and mutant BCR-ABL, including T315I, acting as a pan-BCR-ABL inhibitor. Here, we undertook a combined crystallographic and structure-activity relationship analysis on ponatinib to understand this unique profile. While the ethynyl linker is a key inhibitor functionality that interacts with the gatekeeper, virtually all other components of ponatinib play an essential role in its T315I inhibitory activity. The extensive network of optimized molecular contacts found in the DFG-out binding mode leads to high potency and renders binding less susceptible to disruption by single point mutations. The inhibitory mechanism exemplified by ponatinib may have broad relevance to designing inhibitors against other kinases with mutated gatekeeper residues.

  9. Drug resistance in trypanosomes; effects of metabolic inhibitors, ph and oxidation-reduction potential on normal and resistant trypanosoma rhodesiense

    PubMed Central

    Williamson, J.

    1959-01-01

    A wide variety of metabolic inhibitors tested in vitro for trypanocidal activity on normal and drug-resistant strains of Trypanosoma rhodesiense showed no relation between acquired drug resistance and changes in specific enzymatic function. Oxidation-reduction potential is an important factor in trypanocidal action but is not obviously related to the development of resistance. The dependence on pH of the trypanocidal action of ionizing drugs against both normal and resistant trypanosomes supports the postulate that the development of resistance involves physical changes in cell structures associated with the uptake of drug. PMID:13844959

  10. EGF Induced RET Inhibitor Resistance in CCDC6-RET Lung Cancer Cells

    PubMed Central

    Sung, Ji Hea; Moon, Sung Ung; Kim, Han-Soo; Kim, Jin Won; Lee, Jong Seok

    2017-01-01

    Purpose Rearrangement of the proto-oncogene rearranged during transfection (RET) has been newly identified potential driver mutation in lung adenocarcinoma. Clinically available tyrosine kinase inhibitors (TKIs) target RET kinase activity, which suggests that patients with RET fusion genes may be treatable with a kinase inhibitor. Nevertheless, the mechanisms of resistance to these agents remain largely unknown. Thus, the present study aimed to determine whether epidermal growth factor (EGF) and hepatocyte growth factor (HGF) trigger RET inhibitor resistance in LC-2/ad cells with CCDC6-RET fusion genes. Materials and Methods The effects of EGF and HGF on the susceptibility of a CCDC6-RET lung cancer cell line to RET inhibitors (sunitinib, E7080, vandetanib, and sorafenib) were examined. Results CCDC6-RET lung cancer cells were highly sensitive to RET inhibitors. EGF activated epidermal growth factor receptor (EGFR) and triggered resistance to sunitinib, E7080, vandetanib, and sorafenib by transducing bypass survival signaling through ERK and AKT. Reversible EGFR-TKI (gefitinib) resensitized cancer cells to RET inhibitors, even in the presence of EGF. Endothelial cells, which are known to produce EGF, decreased the sensitivity of CCDC6-RET lung cancer cells to RET inhibitors, an effect that was inhibited by EGFR small interfering RNA (siRNA), anti-EGFR antibody (cetuximab), and EGFR-TKI (Iressa). HGF had relatively little effect on the sensitivity to RET inhibitors. Conclusion EGF could trigger resistance to RET inhibition in CCDC6-RET lung cancer cells, and endothelial cells may confer resistance to RET inhibitors by EGF. E7080 and other RET inhibitors may provide therapeutic benefits in the treatment of RET-positive lung cancer patients. PMID:27873490

  11. Reversing drug resistance of cisplatin by hsp90 inhibitors in human ovarian cancer cells

    PubMed Central

    Zhang, Zhengmao; Xie, Zhen; Sun, Guangyu; Yang, Pingfang; Li, Jia; Yang, Hongfang; Xiao, Shuang; Liu, Yang; Qiu, Hongbing; Qin, Lijun; Zhang, Chao; Zhang, Fenghua; Shan, Baoen

    2015-01-01

    Objective: To investigate the mechanisms for reversing drug resistance of cisplatin (DDP) by Hsp90 inhibitors (geldanamycin (GA), 17-AAG, 17-DMAG) in human ovarian cancer. Methods: Cell proliferation rate in DDP resistant human ovarian cancer cell line SKOV3/DDP and its parent cell line SKOV3 after treatment with Hsp90 inhibitors and/or DDP were tested by MTT assay, and the reversing fold (RF) of DDP by Hsp90 inhibitors was calculated. Cell cycle and cell apoptosis status after treatment were analyzed by flow cytometry. The expression of multiple drug resistance related genes was analyzed by RT-PCR and Western-blot. Results: All three tested Hsp90 inhibitors synergistically inhibited the cell proliferation of SKOV3 with DDP and enhanced the sensitivity of SKOV3/DDP cells to DDP. The RF of DDP by Hsp90 inhibitors were all more than two fold. GA caused cell cycle arrest in G2/M phasein SKOV3 cells. 17-AAG increased cell apoptosis but did not change cell cycle in SKOV3/DDP cells. The mRNA and protein expression levels of various drug resistant related genes including LRP, GST-π, p53, bcl-2, survivin, ERCC1, XRCC1, BRCA1 and BRCA2 were more dramatically altered by Hsp90 inhibitors and DDP in combination compared to Hsp90 inhibitors or DDP treatment alone. Conclusions: Exposure of SKOV3/DDP cells to Hsp90 inhibitors and DDP in combination results in synergistic cytotoxic and pro-apoptotic effects. Hsp90 inhibitors reverse the drug resistance of SKOV3/DDP cells to DDP by modifying the expression of multiple drug resistance related genes. PMID:26221207

  12. MR 20492 and MR 20494: two indolizinone derivatives that strongly inhibit human aromatase.

    PubMed

    Auvray, P; Sourdaine, P; Moslemi, S; Séralini, G E; Sonnet, P; Enguehard, C; Guillon, J; Dallemagne, P; Bureau, R; Rault, S

    1999-01-01

    In this study, we describe the synthesis of a new family of indolizinone derivatives designed to fit an extrahydrophobic pocket within the active site of aromatase and to strongly inhibit human aromatase. This could help improve the specificity of the inhibitors. Equine aromatase, very well characterized biochemically, is used as a comparative model. Indeed, in a previous comparison between both human and equine aromatases, we described the importance of the interaction between the inhibitor and this pocket for the indane derivative MR 20814. MR 20492 and MR 20494 are more potent inhibitors of human aromatase (Ki/Km: 1.0+/-0.3 and 0.5+/-0.3, respectively). The Ki/Km for MR 20494 is slightly higher than that obtained for fadrozole (0.1+/-0.0) and Ki/Km for both indolizinone derivatives are lower than those obtained for 4-hydroxyandrostenedione (1.9+/-0.8) and MR 20814 (8.1+/-.7). These new compounds are not enzyme inactivators. Moreover, as indicated by the higher Ki/Km values obtained with equine enzyme (9.0+/-0.6 and 6.1+/-1.6 for MR 20492 and MR 20494, respectively), both human and equine aromatase active sites appear to be structurally different. Difference absorption spectra study (350-500 nm) revealed that MR20492 and MR20494 were characterized by a combination of type-I and -II spectra with both enzymes. This result could be due to the isomerization of the molecule in polar solvent (Z and E forms). The evaluation of these new molecules, as well as 4-hydroxyandrostenedione and fadrozole, on aromatase activity in transfected 293 cell cultures evidenced a strong inhibition (IC50: 0.20+/-0.03 microM, 0.20+/-0.02 microM and 0.50+/-0.40 microM for MR 20494, fadrozole and 4-OHA, respectively) except for MR 20492 (3.9+/-0.9 microM) and MR 20814 (10.5+/-0.6 microM). These results proved that these molecules formed part of a promising family of potent inhibitors and that they penetrate 293 cells, without evidencing any cytotoxicity in Hela cells with MTT assay. This is

  13. Computational mutation scanning and drug resistance mechanisms of HIV-1 protease inhibitors.

    PubMed

    Hao, Ge-Fei; Yang, Guang-Fu; Zhan, Chang-Guo

    2010-07-29

    The drug resistance of various clinically available HIV-1 protease inhibitors has been studied using a new computational protocol, that is, computational mutation scanning (CMS), leading to valuable insights into the resistance mechanisms and structure-resistance correction of the HIV-1 protease inhibitors associated with a variety of active site and nonactive site mutations. By using the CMS method, the calculated mutation-caused shifts of the binding free energies linearly correlate very well with those derived from the corresponding experimental data, suggesting that the CMS protocol may be used as a generalized approach to predict drug resistance associated with amino acid mutations. Because it is essentially important for understanding the structure-resistance correlation and for structure-based drug design to develop an effective computational protocol for drug resistance prediction, the reasonable and computationally efficient CMS protocol for drug resistance prediction should be valuable for future structure-based design and discovery of antiresistance drugs in various therapeutic areas.

  14. Co-delivery of multiple drug resistance inhibitors by polymer/inorganic hybrid nanoparticles to effectively reverse cancer drug resistance.

    PubMed

    Wu, Cong; Gong, Meng-Qing; Liu, Bo-Ya; Zhuo, Ren-Xi; Cheng, Si-Xue

    2017-01-01

    To effectively reverse multiple drug resistance (MDR) in tumor treatments, a functional nano-sized drug delivery system with active targeting function and pH sensitivity was prepared for the co-delivery of multiple drug resistance inhibitors. Buthionine sulfoximine (BSO) to inhibit GSH synthesis and celecoxib (CXB) to down-regulate P-gp expression were co-loaded in polymer/inorganic hybrid nanoparticles to form buthionine sulfoximine/celecoxib@biotin-heparin/heparin/calcium carbonate/calcium phosphate nanoparticles (BSO/CXB@BNP). To investigate the reversal of MDR, the drug resistant cells (MCF-7/ADR) were pretreated by the dual-inhibitor loaded nanoparticles (BSO/CXB@BNP) followed by the treatment of doxorubicin (DOX) loaded nanoparticles (DOX@BNP). The dual-inhibitor loaded nanoparticles (BSO/CXB@BNP) exhibited greatly enhanced efficiency in down-regulation of GSH and P-gp since BSO and CXB had combined effects on the reduction of GSH and P-gp in drug resistant tumor cells. As a result, BSO/CXB@BNP exhibited a significantly improved capability in reversal of MDR compared with mono-inhibitor loaded nanoparticles (CXB@BNP and BSO@BNP). As compared with free drug resistance inhibitors, delivery of drug resistance inhibitors by functional nanocarriers could obviously improve the therapeutic efficiency due to enhanced cellular uptake and increased intracellular drug accumulation. The study on immunostimulatory effects of different treatments showed that BSO/CXB@BNP treatment resulted in the lowest concentration of interleukin 10, a cytokine related to tumor development. These results suggest the nanoparticulate drug delivery platform developed in this study has promising applications in multiple drug delivery to overcome drug resistance in tumor treatments.

  15. Design of HIV Protease Inhibitors Targeting Protein Backbone: An Effective Strategy for Combating Drug Resistance

    SciTech Connect

    Ghosh, Arun K.; Chapsal, Bruno D.; Weber, Irene T.; Mitsuya, Hiroaki

    2008-06-03

    The discovery of human immunodeficiency virus (HIV) protease inhibitors (PIs) and their utilization in highly active antiretroviral therapy (HAART) have been a major turning point in the management of HIV/acquired immune-deficiency syndrome (AIDS). However, despite the successes in disease management and the decrease of HIV/AIDS-related mortality, several drawbacks continue to hamper first-generation protease inhibitor therapies. The rapid emergence of drug resistance has become the most urgent concern because it renders current treatments ineffective and therefore compels the scientific community to continue efforts in the design of inhibitors that can efficiently combat drug resistance.

  16. Aromatase gene expression in the stallion.

    PubMed

    Lemazurier, E; Sourdaine, P; Nativelle, C; Plainfossé, B; Séralini, G

    2001-06-10

    Adult stallion secretes very high estrogen levels in its testicular vein and semen, and the responsible enzyme cytochrome P450 aromatase (P450 arom) is known to be present mainly in Leydig cells. We studied in further details the distribution of equine aromatase in various adult tissues including the brain (hypothalamic area), liver, kidney, small intestine, muscle, bulbourethral gland and testes. The aromatase mRNA was essentially detected by RT-PCR in testis (169+/-14 amol of aromatase mRNA per microg of total RNA) and was barely detectable in brain, or below 0.1 amol/microg RNA in other tissues. This range of expression was confirmed by ELISA (50+/-7 pg/microg total protein) in the testis, and by immunoblot, evidencing a 53 kDA specific protein band in testis and brain only. The corresponding aromatase activity was well detected, by 3H(2)O release from 1beta, 2beta(3)H-androstenedione, in testis and brain (200+/-23 and 25+/-6 pmol/min per mg, respectively) and below 3 pmol product formed/min per mg in other tissues. This study indicates that the testis, among the tissues analyzed, is the major source of aromatase in the adult stallion, and that the aromatase gene expression is specifically enhanced at this level, and is responsible for the high estrogen synthesis observed. Moreover, the study of aromatase in one colt testis has shown lower levels of transcripts, protein and enzyme activity, evidencing that aromatase is regulated during the development and may serve as a useful marker of testicular function. As the second organ where aromatase mRNA and activity are both well detected is brain, this study also underlines the possible role of neurosteroids in stallion on behaviour, brain function or central endocrine control.

  17. Inhibition of human estrogen synthetase (aromatase) by flavones.

    PubMed

    Kellis, J T; Vickery, L E

    1984-09-07

    Several naturally occurring and synthetic flavones were found to inhibit the aromatization of androstenedione and testosterone to estrogens catalyzed by human placental and ovarian microsomes. These flavones include (in order of decreasing potency) 7,8-benzoflavone, chrysin, apigenin, flavone, flavanone, and quercetin; 5,6-benzoflavone was not inhibitory. 7,8-Benzoflavone and chrysin were potent competitive inhibitors and induced spectral changes in the aromatase cytochrome P-450 indicative of substrate displacement. Flavones may thus compete with steroids in their interaction with certain monooxygenases and thereby alter steroid hormone metabolism.

  18. Aromatase overexpression induces malignant changes in estrogen receptor α negative MCF-10A cells.

    PubMed

    Wang, J; Gildea, J J; Yue, W

    2013-10-31

    Estrogen is a risk factor of breast cancer. Elevated expression of aromatase (estrogen synthase) in breast tissues increases local estradiol concentrations and is associated with breast cancer development, but the causal relationship between aromatase and breast cancer has not been identified. Accumulating data suggest that both estrogen receptor (ER)-dependent and -independent effects are involved in estrogen carcinogenesis. We established a model by expressing aromatase in ERα- MCF-10A human breast epithelial cells to investigate ERα-independent effects of estrogen in the process of malignant transformation. Overexpression of aromatase significantly increased anchorage-independent growth. Parental- or vector-expressing MCF-10A cells did not form colonies under the same conditions. The anchorage-independent growth of MCF-10A(arom) cells can be completely abolished by pre-treatment with the aromatase inhibitor, letrozole. Neither MCF-10A(arom) nor MCF-10A(vector) cells grown in monolayer were affected by short-term exposure to estradiol. Enhanced motility is another characteristic of cellular transformation. Motility of MCF-10A(arom) cells was increased, which could be inhibited by letrozole. Increases in stem cell population in breast cancer tissues are associated with tumor recurrence and metastasis. CD44(high)/CD24(low) is a stem cell marker. We found that CD24 mRNA levels were reduced in MCF-10A(arom) cells compared with those in parental- and vector-transfected cells. By examining individual clones of MCF-10A(arom) with various aromatase activities, we found that the CD24 mRNA levels were inversely correlated with aromatase activity. The ability of MCF-10A(arom) cells to form mammospheres in the absence of serum was increased. Our results suggest that overexpression of aromatase in MCF-10A cells causes malignant transformation. Estrogen metabolite-mediated genotoxicity and induction of a stem cell/progenitor cell population are possible mechanisms. These

  19. Effect and Safety of TNF Inhibitors in Immunoglobulin-Resistant Kawasaki Disease: a Meta-analysis.

    PubMed

    Xue, Li-Jun; Wu, Rong; Du, Gui-Lian; Xu, Yan; Yuan, Kang-Yan; Feng, Zhi-Chun; Pan, Yu-Lin; Hu, Guang-Yu

    2016-08-23

    Previous studies showed that tumor necrosis factor (TNF) inhibitors might decrease the rate of coronary artery abnormalities in pediatrics with intravenous immunoglobulin (IVIG)-resistant Kawasaki disease (KD). Therefore, we aimed to evaluate the effect and safety of TNF inhibitors in IVIG-resistant KD. We undertook a meta-analysis of clinical trials identified in systematic searches of PubMed, EMBASE, Cochrane Database, and Google scholar through May 2016. Five studies were included. Overall, rate of coronary artery aneurysm was comparable between groups (relative risk (RR), 1.05; 95 % confidence interval (95 % CI), 0.60 to 1.81; P = 0.87). No significant differences were recorded between groups in coronary artery Z scores (standardized mean difference (SMD), 0.27; 95 % CI, -0.30 to 0.85; P = 0.35). Meanwhile, TNF inhibitors were not associated with a significant decreased risk of treatment resistance compared with IVIG treatment (RR, 0.65; 95 % CI, 0.37 to 0.15; P = 0.14). However, days of fever was significantly reduced in the TNF inhibitor group (SMD, -0.66; 95 % CI, -0.90 to -0.41; P < 0.001). Additionally, risk of serious adverse events was similar between groups. Therefore, TNF inhibitors could shorten the duration of fever in IVIG-resistant KD. However, TNF inhibitors appear to have no cardioprotective effect in patients with IVIG-resistant KD.

  20. Analysis of putative inhibitors of anthelmintic resistance mechanisms in cattle gastrointestinal nematodes.

    PubMed

    AlGusbi, Salha; Krücken, Jürgen; Ramünke, Sabrina; von Samson-Himmelstjerna, Georg; Demeler, Janina

    2014-08-01

    Effects of the cytochrome P450 inhibitor piperonyl butoxide and the P-glycoprotein inhibitor verapamil on the efficacy of ivermectin and thiabendazole were studied in vitro in susceptible and resistant isolates of the cattle parasitic nematodes Cooperia oncophora and Ostertagia ostertagi. The effects of combined use of drug and piperonyl butoxide/verapamil, respectively, were investigated in the Egg Hatch Assay, the Larval Development Assay and the Larval Migration Inhibition Assay. The effects of piperonyl butoxide and verapamil as inhibitors of thiabendazole and ivermectin responses were particularly marked for larval development, where both inhibitors were able to completely eliminate all differences between susceptible and resistant isolates. Even the lowest concentrations of anthelmintics used in combination with inhibitors caused complete inhibition of development. Differences and/or similarities among responses in different isolates were only obtained in the two other assays: in the Egg Hatch Assay piperonyl butoxide caused a shift in concentration-response curves obtained with thiabendazole to the left for all isolates tested, changing relative differences between isolates. In contrast, an effect of verapamil in the Egg Hatch Assay was only apparent for benzimidazole-resistant isolates. In the Larval Migration Inhibition Assay only ivermectin was tested and piperonyl butoxide shifted the concentration-response curves for all isolates to the left, again eliminating differences in EC50 values between susceptible and resistant isolates. This was not the case using verapamil as an inhibitor, where curves for both susceptible and benzimidazole-resistant isolates shifted to the left in Ostertagia isolates. In Cooperia the picture was more complex with ivermectin-resistant isolates showing a larger shift than the susceptible isolate. Single nucleotide polymorphisms in the β-tubulin isotype 1 gene were investigated. Significantly increased frequencies of

  1. Integrated approach to explore the mechanisms of aromatase inhibition and recovery in fathead minnows (Pimephales promelas).

    PubMed

    Garcia-Reyero, Natàlia; Ekman, Drew R; Habib, Tanwir; Villeneuve, Daniel L; Collette, Timothy W; Bencic, David C; Ankley, Gerald T; Perkins, Edward J

    2014-07-01

    Aromatase, a member of the cytochrome P450 superfamily, is a key enzyme in estradiol synthesis that catalyzes the aromatization of androgens into estrogens in ovaries. Here, we used an integrated approach to assess the mechanistic basis of the direct effects of aromatase inhibition, as well as adaptation and recovery processes in fish. We exposed female fathead minnows (Pimephales promelas) via the water to 30 μg/L of a model aromatase inhibitor, fadrozole, during 8 days (exposure phase). Fish were then held in clean water for 8 more days (recovery phase). Samples were collected at 1, 2, 4, and 8 days of both the exposure and the recovery phases. Transcriptomics, metabolomics, and network inference were used to understand changes and infer connections at the transcript and metabolite level in the ovary. Apical endpoints directly indicative of endocrine function, such as plasma estradiol, testosterone, and vitellogenin levels were also measured. An integrated analysis of the data revealed changes in gene expression consistent with increased testosterone in fadrozole-exposed ovaries. Metabolites such as glycogen and taurine were strongly correlated with increased testosterone levels. Comparison of in vivo and ex vivo steroidogenesis data suggested the accumulation of steroidogenic enzymes, including aromatase, as a mechanism to compensate for aromatase inhibition.

  2. Aromatase inhibition exacerbates pain and reactive gliosis in the dorsal horn of the spinal cord of female rats caused by spinothalamic tract injury.

    PubMed

    Ghorbanpoor, Samar; Garcia-Segura, Luis Miguel; Haeri-Rohani, Ali; Khodagholi, Fariba; Jorjani, Masoumeh

    2014-11-01

    Central pain syndrome is characterized by severe and excruciating pain resulting from a lesion in the central nervous system. Previous studies have shown that estradiol decreases pain and that inhibitors of the enzyme aromatase, which synthesizes estradiol from aromatizable androgens, increases pain sensitivity. In this study we have assessed whether aromatase expression in the dorsal horns of the spinal cord is altered in a rat model of central pain syndrome, induced by the unilateral electrolytic lesion of the spinothalamic tract. Protein and mRNA levels of aromatase, as well as the protein and mRNA levels of estrogen receptors α and β, were increased in the dorsal horn of female rats after spinothalamic tract injury, suggesting that the injury increased estradiol synthesis and signaling in the dorsal horn. To determine whether the increased aromatase expression in this pain model may participate in the control of pain, mechanical allodynia thresholds were determined in both hind paws after the intrathecal administration of letrozole, an aromatase inhibitor. Aromatase inhibition enhanced mechanical allodynia in both hind paws. Because estradiol is known to regulate gliosis we assessed whether the spinothalamic tract injury and aromatase inhibition regulated gliosis in the dorsal horn. The proportion of microglia with a reactive phenotype and the number of glial fibrillary acidic protein-immunoreactive astrocytes were increased by the injury in the dorsal horn. Aromatase inhibition enhanced the effect of the injury on gliosis. Furthermore, a significant a positive correlation of mechanical allodynia and gliosis in the dorsal horn was detected. These findings suggest that aromatase is up-regulated in the dorsal horn in a model of central pain syndrome and that aromatase activity in the spinal cord reduces mechanical allodynia by controlling reactive gliosis in the dorsal horn.

  3. Potent Inhibitors of Acetyltransferase Eis Overcome Kanamycin Resistance in Mycobacterium tuberculosis.

    PubMed

    Willby, Melisa J; Green, Keith D; Gajadeera, Chathurada S; Hou, Caixia; Tsodikov, Oleg V; Posey, James E; Garneau-Tsodikova, Sylvie

    2016-06-17

    A major cause of tuberculosis (TB) resistance to the aminoglycoside kanamycin (KAN) is the Mycobacterium tuberculosis (Mtb) acetyltransferase Eis. Upregulation of this enzyme is responsible for inactivation of KAN through acetylation of its amino groups. A 123 000-compound high-throughput screen (HTS) yielded several small-molecule Eis inhibitors that share an isothiazole S,S-dioxide heterocyclic core. These were investigated for their structure-activity relationships. Crystal structures of Eis in complex with two potent inhibitors show that these molecules are bound in the conformationally adaptable aminoglycoside binding site of the enzyme, thereby obstructing binding of KAN for acetylation. Importantly, we demonstrate that several Eis inhibitors, when used in combination with KAN against resistant Mtb, efficiently overcome KAN resistance. This approach paves the way toward development of novel combination therapies against aminoglycoside-resistant TB.

  4. Azole fungicides affect mammalian steroidogenesis by inhibiting sterol 14 alpha-demethylase and aromatase.

    PubMed

    Zarn, Jürg A; Brüschweiler, Beat J; Schlatter, Josef R

    2003-03-01

    Azole compounds play a key role as antifungals in agriculture and in human mycoses and as non-steroidal antiestrogens in the treatment of estrogen-responsive breast tumors in postmenopausal women. This broad use of azoles is based on their inhibition of certain pathways of steroidogenesis by high-affinity binding to the enzymes sterol 14-alpha-demethylase and aromatase. Sterol 14-alpha-demethylase is crucial for the production of meiosis-activating sterols, which recently were shown to modulate germ cell development in both sexes of mammals. Aromatase is responsible for the physiologic balance of androgens and estrogens. At high doses, azole fungicides and other azole compounds affect reproductive organs, fertility, and development in several species. These effects may be explained by inhibition of sterol 14-alpha-demethylase and/or aromatase. In fact, several azole compounds were shown to inhibit these enzymes in vitro, and there is also strong evidence for inhibiting activity in vivo. Furthermore, the specificity of the enzyme inhibition of several of these compounds is poor, both with respect to fungal versus nonfungal sterol 14-alpha-demethylases and versus other P450 enzymes including aromatase. To our knowledge, this is the first review on sterol 14-alpha-demethylase and aromatase as common targets of azole compounds and the consequence for steroidogenesis. We conclude that many azole compounds developed as inhibitors of fungal sterol 14-alpha-demethylase are inhibitors also of mammalian sterol 14-alpha-demethylase and mammalian aromatase with unknown potencies. For human health risk assessment, data on comparative potencies of azole fungicides to fungal and human enzymes are needed.

  5. Azole fungicides affect mammalian steroidogenesis by inhibiting sterol 14 alpha-demethylase and aromatase.

    PubMed Central

    Zarn, Jürg A; Brüschweiler, Beat J; Schlatter, Josef R

    2003-01-01

    Azole compounds play a key role as antifungals in agriculture and in human mycoses and as non-steroidal antiestrogens in the treatment of estrogen-responsive breast tumors in postmenopausal women. This broad use of azoles is based on their inhibition of certain pathways of steroidogenesis by high-affinity binding to the enzymes sterol 14-alpha-demethylase and aromatase. Sterol 14-alpha-demethylase is crucial for the production of meiosis-activating sterols, which recently were shown to modulate germ cell development in both sexes of mammals. Aromatase is responsible for the physiologic balance of androgens and estrogens. At high doses, azole fungicides and other azole compounds affect reproductive organs, fertility, and development in several species. These effects may be explained by inhibition of sterol 14-alpha-demethylase and/or aromatase. In fact, several azole compounds were shown to inhibit these enzymes in vitro, and there is also strong evidence for inhibiting activity in vivo. Furthermore, the specificity of the enzyme inhibition of several of these compounds is poor, both with respect to fungal versus nonfungal sterol 14-alpha-demethylases and versus other P450 enzymes including aromatase. To our knowledge, this is the first review on sterol 14-alpha-demethylase and aromatase as common targets of azole compounds and the consequence for steroidogenesis. We conclude that many azole compounds developed as inhibitors of fungal sterol 14-alpha-demethylase are inhibitors also of mammalian sterol 14-alpha-demethylase and mammalian aromatase with unknown potencies. For human health risk assessment, data on comparative potencies of azole fungicides to fungal and human enzymes are needed. PMID:12611652

  6. The pan-PI3K inhibitor GDC-0941 activates canonical WNT signaling to confer resistance in TNBC cells: resistance reversal with WNT inhibitor.

    PubMed

    Tzeng, Huey-En; Yang, Lixin; Chen, Kemin; Wang, Yafan; Liu, Yun-Ru; Pan, Shiow-Lin; Gaur, Shikha; Hu, Shuya; Yen, Yun

    2015-05-10

    The pan-PI3K inhibitors are one treatment option for triple-negative breast cancer (TNBC). However, this treatment is ineffective for unknown reasons. Here, we report that aberrant expression of wingless-type MMTV integration site family (WNT) and activated WNT signals, which crosstalk with the PI3K-AKT-mTOR signaling pathway through GSK3β, plays the most critical role in resistance to pan-PI3K inhibitors in TNBC cells. GDC-0941 is a pan-PI3K inhibitor that activates the WNT/beta-catenin pathway in TNBC cells through stimulation of WNT secretion. GDC-0941-triggered WNT/beta-catenin pathway activation was observed in MDA-MB-231 and HCC1937 cells, which are TNBC cell lines showing aberrant WNT/beta-catenin activation, and not in SKBR3 and MCF7 cells. This observation is further investigated in vivo. GDC-0941 exhibited minimal tumor inhibition in MDA-MB-231 cells, but it significantly suppressed tumor growth in HER-positive SK-BR3 cells. In vivo mechanism study revealed the activation of WNT/beta-catenin pathway by GDC-0941. A synergistic effect was observed when combined treatment with GDC-0941 and the WNT inhibitor LGK974 at low concentrations in MDA-MB-231 cells. These findings indicated that WNT pathway activation conferred resistance in TNBC cells treated with GDC-0941. This resistance may be further circumvented through combined treatment with pan-PI3K and WNT inhibitors. Future clinical trials of these two inhibitors are warranted.

  7. Structural Basis of Resistance to Anti-Cytochrome bc1 Complex Inhibitors: Implication for Drug Improvement

    PubMed Central

    Esser, Lothar; Yu, Chang-An; Xia, Di

    2016-01-01

    The emergence of drug resistance has devastating economic and social consequences, a testimonial of which is the rise and fall of inhibitors against the respiratory component cytochrome bc1 complex, a time tested and highly effective target for disease control. Unfortunately, the mechanism of resistance is a multivariate problem, including primarily mutations in the gene of the cytochrome b subunit but also activation of alternative pathways of ubiquinol oxidation and pharmacokinetic effects. There is a considerable interest in designing new bc1 inhibitors with novel modes of binding and lower propensity to induce the development of resistance. The accumulation of crystallographic data of bc1 complexes with and without inhibitors bound provides the structural basis for rational drug design. In particular, the cytochrome b subunit offers two distinct active sites that can be targeted for inhibition - the quinol oxidation site and the quinone reduction site. This review brings together available structural information of inhibited bc1 by various quinol oxidation- and reduction-site inhibitors, the inhibitor binding modes, conformational changes upon inhibitor binding of side chains in the active site and large scale domain movements of the iron-sulfur protein subunit. Structural data analysis provides a clear understanding of where and why existing inhibitors fail and points towards promising alternatives. PMID:23688079

  8. Molecular Characterization of Clinical Isolates of Human Immunodeficiency Virus Resistant to the Protease Inhibitor Darunavir

    SciTech Connect

    Sasková, Klára Grantz; Koíek, Milan; Rezácová, Pavlína; Brynda, Jirí; Yashina, Tatyana; Kagan, Ron M.; Konvalinka, Jan

    2010-03-04

    Darunavir is the most recently approved human immunodeficiency virus (HIV) protease (PR) inhibitor (PI) and is active against many HIV type 1 PR variants resistant to earlier-generation PIs. Darunavir shows a high genetic barrier to resistance development, and virus strains with lower sensitivity to darunavir have a higher number of PI resistance-associated mutations than viruses resistant to other PIs. In this work, we have enzymologically and structurally characterized a number of highly mutated clinically derived PRs with high levels of phenotypic resistance to darunavir. With 18 to 21 amino acid residue changes, the PR variants studied in this work are the most highly mutated HIV PR species ever studied by means of enzyme kinetics and X-ray crystallography. The recombinant proteins showed major defects in substrate binding, while the substrate turnover was less affected. Remarkably, the overall catalytic efficiency of the recombinant PRs (5% that of the wild-type enzyme) is still sufficient to support polyprotein processing and particle maturation in the corresponding viruses. The X-ray structures of drug-resistant PRs complexed with darunavir suggest that the impaired inhibitor binding could be explained by change in the PR-inhibitor hydrogen bond pattern in the P2 binding pocket due to a substantial shift of the aminophenyl moiety of the inhibitor. Recombinant virus phenotypic characterization, enzyme kinetics, and X-ray structural analysis thus help to explain darunavir resistance development in HIV-positive patients.

  9. Coevolutionary analysis of resistance-evading peptidomimetic inhibitors of HIV-1 protease.

    PubMed

    Rosin, C D; Belew, R K; Morris, G M; Olson, A J; Goodsell, D S

    1999-02-16

    We have developed a coevolutionary method for the computational design of HIV-1 protease inhibitors selected for their ability to retain efficacy in the face of protease mutation. For HIV-1 protease, typical drug design techniques are shown to be ineffective for the design of resistance-evading inhibitors: An inhibitor that is a direct analogue of one of the natural substrates will be susceptible to resistance mutation, as will inhibitors designed to fill the active site of the wild-type or a mutant enzyme. Two design principles are demonstrated: (i) For enzymes with broad substrate specificity, such as HIV-1 protease, resistance-evading inhibitors are best designed against the immutable properties of the active site-the properties that must be conserved in any mutant protease to retain the ability to bind and cleave all of the native substrates. (ii) Robust resistance-evading inhibitors can be designed by optimizing activity simultaneously against a large set of mutant enzymes, incorporating as much of the mutational space as possible.

  10. Coevolutionary analysis of resistance-evading peptidomimetic inhibitors of HIV-1 protease

    PubMed Central

    Rosin, Christopher D.; Belew, Richard K.; Morris, Garrett M.; Olson, Arthur J.; Goodsell, David S.

    1999-01-01

    We have developed a coevolutionary method for the computational design of HIV-1 protease inhibitors selected for their ability to retain efficacy in the face of protease mutation. For HIV-1 protease, typical drug design techniques are shown to be ineffective for the design of resistance-evading inhibitors: An inhibitor that is a direct analogue of one of the natural substrates will be susceptible to resistance mutation, as will inhibitors designed to fill the active site of the wild-type or a mutant enzyme. Two design principles are demonstrated: (i) For enzymes with broad substrate specificity, such as HIV-1 protease, resistance-evading inhibitors are best designed against the immutable properties of the active site—the properties that must be conserved in any mutant protease to retain the ability to bind and cleave all of the native substrates. (ii) Robust resistance-evading inhibitors can be designed by optimizing activity simultaneously against a large set of mutant enzymes, incorporating as much of the mutational space as possible. PMID:9990030

  11. Identification and characterization of influenza variants resistant to a viral endonuclease inhibitor

    SciTech Connect

    Song, Min-Suk; Kumar, Gyanendra; Shadrick, William R.; Zhou, Wei; Jeevan, Trushar; Li, Zhenmei; Slavish, P. Jake; Fabrizio, Thomas P.; Yoon, Sun-Woo; Webb, Thomas R.; Webby, Richard J.; White, Stephen W.

    2016-03-14

    The influenza endonuclease is an essential subdomain of the viral RNA polymerase. It processes host pre-mRNAs to serve as primers for viral mRNA and is an attractive target for antiinfluenza drug discovery. Compound L-742,001 is a prototypical endonuclease inhibitor, and we found that repeated passaging of influenza virus in the presence of this drug did not lead to the development of resistant mutant strains. Reduced sensitivity to L-742,001 could only be induced by creating point mutations via a random mutagenesis strategy. These mutations mapped to the endonuclease active site where they can directly impact inhibitor binding. Engineered viruses containing the mutations showed resistance to L-742,001 both in vitro and in vivo, with only a modest reduction in fitness. Introduction of the mutations into a second virus also increased its resistance to the inhibitor. Using the isolated wild-type and mutant endonuclease domains, we used kinetics, inhibitor binding and crystallography to characterize how the two most significant mutations elicit resistance to L-742,001. These studies lay the foundation for the development of a new class of influenza therapeutics with reduced potential for the development of clinical endonuclease inhibitor-resistant influenza strains.

  12. Identification and characterization of influenza variants resistant to a viral endonuclease inhibitor

    DOE PAGES

    Song, Min-Suk; Kumar, Gyanendra; Shadrick, William R.; ...

    2016-03-14

    The influenza endonuclease is an essential subdomain of the viral RNA polymerase. It processes host pre-mRNAs to serve as primers for viral mRNA and is an attractive target for antiinfluenza drug discovery. Compound L-742,001 is a prototypical endonuclease inhibitor, and we found that repeated passaging of influenza virus in the presence of this drug did not lead to the development of resistant mutant strains. Reduced sensitivity to L-742,001 could only be induced by creating point mutations via a random mutagenesis strategy. These mutations mapped to the endonuclease active site where they can directly impact inhibitor binding. Engineered viruses containing themore » mutations showed resistance to L-742,001 both in vitro and in vivo, with only a modest reduction in fitness. Introduction of the mutations into a second virus also increased its resistance to the inhibitor. Using the isolated wild-type and mutant endonuclease domains, we used kinetics, inhibitor binding and crystallography to characterize how the two most significant mutations elicit resistance to L-742,001. These studies lay the foundation for the development of a new class of influenza therapeutics with reduced potential for the development of clinical endonuclease inhibitor-resistant influenza strains.« less

  13. Cooperating JAK1 and JAK3 mutants increase resistance to JAK inhibitors.

    PubMed

    Springuel, Lorraine; Hornakova, Tekla; Losdyck, Elisabeth; Lambert, Fanny; Leroy, Emilie; Constantinescu, Stefan N; Flex, Elisabetta; Tartaglia, Marco; Knoops, Laurent; Renauld, Jean-Christophe

    2014-12-18

    The acquisition of growth signal self-sufficiency is 1 of the hallmarks of cancer. We previously reported that the murine interleukin-9-dependent TS1 cell line gives rise to growth factor-independent clones with constitutive activation of the Janus kinase (JAK)- signal transducer and activator of transcription (STAT) pathway. Here, we show that this transforming event results from activating mutations either in JAK1, JAK3, or in both kinases. Transient and stable expression of JAK1 and/or JAK3 mutants showed that each mutant induces STAT activation and that their coexpression further increases this activation. The proliferation of growth factor-independent TS1 clones can be efficiently blocked by JAK inhibitors such as ruxolitinib or CMP6 in short-term assays. However, resistant clones occur upon long-term culture in the presence of inhibitors. Surprisingly, resistance to CMP6 was not caused by the acquisition of secondary mutations in the adenosine triphosphate-binding pocket of the JAK mutant. Indeed, cells that originally showed a JAK1-activating mutation became resistant to inhibitors by acquiring another activating mutation in JAK3, whereas cells that originally showed a JAK3-activating mutation became resistant to inhibitors by acquiring another activating mutation in JAK1. These observations underline the cooperation between JAK1 and JAK3 mutants in T-cell transformation and represent a new mechanism of acquisition of resistance against JAK inhibitors.

  14. Identification and characterization of influenza variants resistant to a viral endonuclease inhibitor

    PubMed Central

    Song, Min-Suk; Kumar, Gyanendra; Shadrick, William R.; Zhou, Wei; Jeevan, Trushar; Li, Zhenmei; Slavish, P. Jake; Yoon, Sun-Woo; Webb, Thomas R.; Webby, Richard J.; White, Stephen W.

    2016-01-01

    The influenza endonuclease is an essential subdomain of the viral RNA polymerase. It processes host pre-mRNAs to serve as primers for viral mRNA and is an attractive target for antiinfluenza drug discovery. Compound L-742,001 is a prototypical endonuclease inhibitor, and we found that repeated passaging of influenza virus in the presence of this drug did not lead to the development of resistant mutant strains. Reduced sensitivity to L-742,001 could only be induced by creating point mutations via a random mutagenesis strategy. These mutations mapped to the endonuclease active site where they can directly impact inhibitor binding. Engineered viruses containing the mutations showed resistance to L-742,001 both in vitro and in vivo, with only a modest reduction in fitness. Introduction of the mutations into a second virus also increased its resistance to the inhibitor. Using the isolated wild-type and mutant endonuclease domains, we used kinetics, inhibitor binding and crystallography to characterize how the two most significant mutations elicit resistance to L-742,001. These studies lay the foundation for the development of a new class of influenza therapeutics with reduced potential for the development of clinical endonuclease inhibitor-resistant influenza strains. PMID:26976575

  15. Combination of a STAT3 Inhibitor and an mTOR Inhibitor Against a Temozolomide-resistant Glioblastoma Cell Line

    PubMed Central

    MIYATA*, HARUO; ASHIZAWA*, TADASHI; IIZUKA, AKIRA; KONDOU, RYOTA; NONOMURA, CHIZU; SUGINO, TAKASHI; URAKAMI, KENICHI; ASAI, AKIRA; HAYASHI, NAKAMASA; MITSUYA, KOICHI; NAKASU, YOKO; YAMAGUCHI, KEN; AKIYAMA, YASUTO

    2016-01-01

    Background: Temozolomide-resistant (TMZ-R) glioblastoma is very difficult to treat, and a novel approach to overcome resistance is needed. Materials and Methods: The efficacy of a combination treatment of STAT3 inhibitor, STX-0119, with rapamycin was investigated against our established TMZ-resistant U87 cell line. Results: The growth-inhibitory effect of the combination treatment was significant against the TMZ-R U87 cell line (IC50: 78 μM for STX-0119, 30.5 μM for rapamycin and 11.3 μM for combination of the two). Western blotting analysis demonstrated that the inhibitory effect of STX-0119 on S6 and 4E-BP1 activation through regulation of YKL-40 expression occurred in addition to the inhibitory effect of rapamycin against the mTOR pathway. Conclusion: These results suggest that the STAT3 pathway is associated with the mTOR downstream pathway mediated by YKL-40 protein, and the combination therapy of the STAT3 inhibitor and rapamycin could be worth developing as a novel therapeutic approach against TMZ-resistant relapsed gliomas. Abbreviations: GB: Glioblastoma, TMZ: temozolomide, MGMT: O6-methylguanine-O6-methylguanine-DNAmethyltransferase, STAT: signal transducer and activator of transcription, mTOR: mammalian target of rapamycin, shRNA: small hairpin RNA. PMID:28031240

  16. Covalent EGFR inhibitor analysis reveals importance of reversible interactions to potency and mechanisms of drug resistance.

    PubMed

    Schwartz, Phillip A; Kuzmic, Petr; Solowiej, James; Bergqvist, Simon; Bolanos, Ben; Almaden, Chau; Nagata, Asako; Ryan, Kevin; Feng, Junli; Dalvie, Deepak; Kath, John C; Xu, Meirong; Wani, Revati; Murray, Brion William

    2014-01-07

    Covalent inhibition is a reemerging paradigm in kinase drug design, but the roles of inhibitor binding affinity and chemical reactivity in overall potency are not well-understood. To characterize the underlying molecular processes at a microscopic level and determine the appropriate kinetic constants, specialized experimental design and advanced numerical integration of differential equations are developed. Previously uncharacterized investigational covalent drugs reported here are shown to be extremely effective epidermal growth factor receptor (EGFR) inhibitors (kinact/Ki in the range 10(5)-10(7) M(-1)s(-1)), despite their low specific reactivity (kinact ≤ 2.1 × 10(-3) s(-1)), which is compensated for by high binding affinities (Ki < 1 nM). For inhibitors relying on reactivity to achieve potency, noncovalent enzyme-inhibitor complex partitioning between inhibitor dissociation and bond formation is central. Interestingly, reversible binding affinity of EGFR covalent inhibitors is highly correlated with antitumor cell potency. Furthermore, cellular potency for a subset of covalent inhibitors can be accounted for solely through reversible interactions. One reversible interaction is between EGFR-Cys797 nucleophile and the inhibitor's reactive group, which may also contribute to drug resistance. Because covalent inhibitors target a cysteine residue, the effects of its oxidation on enzyme catalysis and inhibitor pharmacology are characterized. Oxidation of the EGFR cysteine nucleophile does not alter catalysis but has widely varied effects on inhibitor potency depending on the EGFR context (e.g., oncogenic mutations), type of oxidation (sulfinylation or glutathiolation), and inhibitor architecture. These methods, parameters, and insights provide a rational framework for assessing and designing effective covalent inhibitors.

  17. Field trials for corrosion inhibitor selection and optimization, using a new generation of electrical resistance probes

    SciTech Connect

    Ridd, B.; Blakset, T.J.; Queen, D.

    1998-12-31

    Even with today`s availability of corrosion resistant alloys, carbon steels protected by corrosion inhibitors still dominate the material selection for pipework in the oil and gas production. Even though laboratory screening tests of corrosion inhibitor performance provides valuable data, the real performance of the chemical can only be studied through field trials which provide the ultimate test to evaluate the effectiveness of an inhibitor under actual operating conditions. A new generation of electrical resistance probe has been developed, allowing highly sensitive and immediate response to changes in corrosion rates on the internal environment of production pipework. Because of the high sensitivity, the probe responds to small changes in the corrosion rate, and it provides the corrosion engineer with a highly effective method of optimizing the use of inhibitor chemicals resulting in confidence in corrosion control and minimizing detrimental environmental effects.

  18. Mechanisms of resistance to third-generation EGFR tyrosine kinase inhibitors.

    PubMed

    Wang, Shuhang; Song, Yongping; Yan, Feifei; Liu, Delong

    2016-12-01

    The tyrosine kinase inhibitors (TKI) of the epidermal growth factor receptor (EGFR) are becoming the first line of therapy for advanced non-small cell lung cancer (NSCLC). Acquired mutations in EGFR account for one of the major mechanisms of resistance to the TKIs. Three generations of EGFR TKIs have been used in clinical applications. AZD9291 (osimertinib; Tagrisso) is the first and only FDA approved third-generation EGFR TKI for T790M-positive advanced NSCLC patients. However, resistance to AZD9291 arises after 9-13 months of therapy. The mechanisms of resistance to third-generation inhibitors reported to date include the EGFR C797S mutation, EGFR L718Q mutation, and amplifications of HER-2, MET, or ERBB2. To overcome the acquired resistance to AZD9291, EAI045 was discovered and recently reported to be an allosteric EGFR inhibitor that overcomes T790M- and C797S-mediated resistance. This review summarizes recent investigations on the mechanisms of resistance to the EGFR TKIs, as well as the latest development of EAI045 as a fourth-generation EGFR inhibitor.

  19. Coronaviruses resistant to a 3C-like protease inhibitor are attenuated for replication and pathogenesis, revealing a low genetic barrier but high fitness cost of resistance.

    PubMed

    Deng, Xufang; StJohn, Sarah E; Osswald, Heather L; O'Brien, Amornrat; Banach, Bridget S; Sleeman, Katrina; Ghosh, Arun K; Mesecar, Andrew D; Baker, Susan C

    2014-10-01

    Viral protease inhibitors are remarkably effective at blocking the replication of viruses such as human immunodeficiency virus and hepatitis C virus, but they inevitably lead to the selection of inhibitor-resistant mutants, which may contribute to ongoing disease. Protease inhibitors blocking the replication of coronavirus (CoV), including the causative agents of severe acute respiratory syndrome (SARS) and Middle East respiratory syndrome (MERS), provide a promising foundation for the development of anticoronaviral therapeutics. However, the selection and consequences of inhibitor-resistant CoVs are unknown. In this study, we exploited the model coronavirus, mouse hepatitis virus (MHV), to investigate the genotype and phenotype of MHV quasispecies selected for resistance to a broad-spectrum CoV 3C-like protease (3CLpro) inhibitor. Clonal sequencing identified single or double mutations within the 3CLpro coding sequence of inhibitor-resistant virus. Using reverse genetics to generate isogenic viruses with mutant 3CLpros, we found that viruses encoding double-mutant 3CLpros are fully resistant to the inhibitor and exhibit a significant delay in proteolytic processing of the viral replicase polyprotein. The inhibitor-resistant viruses also exhibited postponed and reduced production of infectious virus particles. Biochemical analysis verified double-mutant 3CLpro enzyme as impaired for protease activity and exhibiting reduced sensitivity to the inhibitor and revealed a delayed kinetics of inhibitor hydrolysis and activity restoration. Furthermore, the inhibitor-resistant virus was shown to be highly attenuated in mice. Our study provides the first insight into the pathogenicity and mechanism of 3CLpro inhibitor-resistant CoV mutants, revealing a low genetic barrier but high fitness cost of resistance. Importance: RNA viruses are infamous for their ability to evolve in response to selective pressure, such as the presence of antiviral drugs. For coronaviruses such as

  20. Novel inhibitors of the methicillin-resistant Staphylococcus aureus (MRSA)-pyruvate kinase.

    PubMed

    El-Sayed, Mardia Telep; Zoraghi, Roya; Reiner, Neil; Suzen, Sibel; Ohlsen, Knut; Lalk, Michael; Altanlar, Nurten; Hilgeroth, Andreas

    2016-12-01

    Novel bisindolyl-cycloalkane indoles resulted from the reaction of aliphatic dialdehydes and indole. As bisindolyl-natural alkaloid compounds have recently been reported as inhibitors of the methicillin-resistant Staphylococcus aureus (MRSA)-pyruvate kinase (PK), we tested our novel compounds as MRSA PK inhibitors and now report first inhibiting activities. We discuss structure-activity relationships of structurally varied compounds. Activity influencing substituents have been characterized and relations to antibacterial activities of the most active compounds have been proved.

  1. Aromatase inhibition remodels the clonal architecture of estrogen-receptor-positive breast cancers

    PubMed Central

    Miller, Christopher A.; Gindin, Yevgeniy; Lu, Charles; Griffith, Obi L; Griffith, Malachi; Shen, Dong; Hoog, Jeremy; Li, Tiandao; Larson, David E.; Watson, Mark; Davies, Sherri R; Hunt, Kelly; Suman, Vera J.; Snider, Jacqueline; Walsh, Thomas; Colditz, Graham A.; DeSchryver, Katherine; Wilson, Richard K.; Mardis, Elaine R.; Ellis, Matthew J.

    2016-01-01

    Resistance to oestrogen-deprivation therapy is common in oestrogen-receptor-positive (ER+) breast cancer. To better understand the contributions of tumour heterogeneity and evolution to resistance, here we perform comprehensive genomic characterization of 22 primary tumours sampled before and after 4 months of neoadjuvant aromatase inhibitor (NAI) treatment. Comparing whole-genome sequencing of tumour/normal pairs from the two time points, with coincident tumour RNA sequencing, reveals widespread spatial and temporal heterogeneity, with marked remodelling of the clonal landscape in response to NAI. Two cases have genomic evidence of two independent tumours, most obviously an ER− ‘collision tumour', which was only detected after NAI treatment of baseline ER+ disease. Many mutations are newly detected or enriched post treatment, including two ligand-binding domain mutations in ESR1. The observed clonal complexity of the ER+ breast cancer genome suggests that precision medicine approaches based on genomic analysis of a single specimen are likely insufficient to capture all clinically significant information. PMID:27502118

  2. Aromatase Activity and Bone Loss in Men

    PubMed Central

    Merlotti, Daniela; Gennari, Luigi; Stolakis, Konstantinos; Nuti, Ranuccio

    2011-01-01

    Aromatase is a specific component of the cytochrome P450 enzyme system responsible for the transformation of androgen precursors into estrogens. This enzyme is encoded by the CYP19A1 gene located at chromosome 15q21.2, that is, expressed in ovary and testis, but also in many extraglandular sites such as the placenta, brain, adipose tissue, and bone. The activity of aromatase regulates the concentrations of estrogens with endocrine, paracrine, and autocrine effects on target issues including bone. Importantly, extraglandular aromatization of circulating androgen precursors is the major source of estrogen in men. Clinical and experimental evidences clearly indicate that aromatase activity and estrogen production are necessary for longitudinal bone growth, the attainment of peak bone mass, pubertal growth spurt, epiphyseal closure, and normal bone remodeling in young individuals. Moreover, with aging, individual differences in aromatase activity may significantly affect bone loss and fracture risk in men. PMID:21772971

  3. Inhibition of aromatase activity by flavonoids.

    PubMed

    Jeong, H J; Shin, Y G; Kim, I H; Pezzuto, J M

    1999-06-01

    In searching for potent cancer chemopreventive agents from synthetic or natural products, 28 randomly selected flavonoids were screened for inhibitory effects against partially purified aromatase prepared from human placenta. Over 50% of the flavonoids significantly inhibited aromatase activity, with greatest activity being demonstrated with apigenin (IC50: 0.9 microg/mL), chrysin (IC50: 1.1 microg/mL), and hesperetin (IC50: 1.0 microg/mL).

  4. Testosterone-derived estradiol production by male endothelium is robust and dependent on p450 aromatase via estrogen receptor alpha.

    PubMed

    Villablanca, Amparo C; Tetali, Sarada; Altman, Robin; Ng, Kenneth F; Rutledge, John C

    2013-12-01

    Vascular endothelium expresses both the estrogen receptors (ERs) α and β, and ERα mediates development of early atherosclerosis in male mice. This process is thought to be testosterone-dependent. We hypothesized that male murine aortic endothelium produces robust levels of estradiol by aromatase conversion of testosterone, and that regulation of this process is mediated by the presence of ERs, primarily ERα. Aortic endothelium was isolated from ERα knockout (ERα -/-) and wild-type (ERα +/+) male mice and treated with testosterone or the 5α reduction product dihydrotestosterone (DHT), with or without the P450 aromatase inhibitor anastrazole, or a non-specific estrogen receptor antagonist. Aromatase gene expression and estradiol production were assayed. Treatment with testosterone, but not DHT, caused increased aromatase expression and estradiol production in ERα +/+ endothelium that was attenuated by disruption of ERα in the ERα -/- group. Anastrazole inhibition of aromatase reduced testosterone-induced aromatase expression and estradiol levels in both ERα -/- and ERα +/+ endothelium. Antagonism of both ERs decreased testosterone-induced aromatase expression in both wild-type and knockout groups. The effects of the receptor antagonist on estradiol production differed between the two groups, however, with a reduction in estradiol release from the ERα +/+ cells and complete abolition of estradiol release from the ERα -/- cells. Thus, estradiol production in vascular endothelium from male mice is robust, depends on the aromatic conversion of testosterone and requires functional ERα to achieve maximal levels of estradiol generation. Local vascular production of aromatase-mediated estradiol in response to circulating testosterone may affect ERα-dependent mechanisms to increase susceptibility to early atheroma formation in male mice. This pathway may have important therapeutic relevance for reducing the risk of atherosclerotic cardiovascular disease in

  5. Zoledronic acid inhibits aromatase activity and phosphorylation: potential mechanism for additive zoledronic acid and letrozole drug interaction.

    PubMed

    Schech, Amanda J; Nemieboka, Brandon E; Brodie, Angela H

    2012-11-01

    Zoledronic acid (ZA), a bisphosphonate originally indicated for use in osteoporosis, has been reported to exert a direct effect on breast cancer cells, although the mechanism of this effect is currently unknown. Data from the ABCSG-12 and ZO-FAST clinical trials suggest that treatment with the combination of ZA and aromatase inhibitors (AI) result in increased disease free survival in breast cancer patients over AI alone. To determine whether the mechanism of this combination involved inhibition of aromatase, AC-1 cells (MCF-7 human breast cancer cells transfected with an aromatase construct) were treated simultaneously with combinations of ZA and AI letrozole. This combination significantly increased inhibition of aromatase activity of AC-1 cells when compared to letrozole alone. Treatment of 1 nM letrozole in combination with 1 μM or 10 μM ZA resulted in an additive drug interaction on inhibition of cell viability, as measured by MTT assay. Treatment with ZA was found to inhibit phosphorylation of aromatase on serine residues. Zoledronic acid was also shown to be more effective in inhibiting cell viability in aromatase transfected AC-1 cells when compared to inhibition of cell viability observed in non-transfected MCF-7. Estradiol was able to partially rescue the effect of 1 μM and 10 μM ZA on cell viability following treatment for 72 h, as shown by a shift to the right in the estradiol dose-response curve. In conclusion, these results indicate that the combination of ZA and letrozole results in an additive inhibition of cell viability. Furthermore, ZA alone can inhibit aromatase activity through inhibition of serine phosphorylation events important for aromatase enzymatic activity and contributes to inhibition of cell viability.

  6. Marine Natural Products as Breast Cancer Resistance Protein Inhibitors

    PubMed Central

    Cherigo, Lilia; Lopez, Dioxelis; Martinez-Luis, Sergio

    2015-01-01

    Breast cancer resistance protein (BCRP) is a protein belonging to the ATP-binding cassette (ABC) transporter superfamily that has clinical relevance due to its multi-drug resistance properties in cancer. BCRP can be associated with clinical cancer drug resistance, in particular acute myelogenous or acute lymphocytic leukemias. The overexpression of BCRP contributes to the resistance of several chemotherapeutic drugs, such as topotecan, methotrexate, mitoxantrone, doxorubicin and daunorubicin. The Food and Drugs Administration has already recognized that BCRP is clinically one of the most important drug transporters, mainly because it leads to a reduction of clinical efficacy of various anticancer drugs through its ATP-dependent drug efflux pump function as well as its apparent participation in drug resistance. This review article aims to summarize the different research findings on marine natural products with BCRP inhibiting activity. In this sense, the potential modulation of physiological targets of BCRP by natural or synthetic compounds offers a great possibility for the discovery of new drugs and valuable research tools to recognize the function of the complex ABC-transporters. PMID:25854646

  7. Probing the origins of aromatase inhibitory activity of disubstituted coumarins via QSAR and molecular docking.

    PubMed

    Worachartcheewan, Apilak; Suvannang, Naravut; Prachayasittikul, Supaluk; Prachayasittikul, Virapong; Nantasenamat, Chanin

    2014-01-01

    This study investigated the quantitative structure-activity relationship (QSAR) of imidazole derivatives of 4,7-disubstituted coumarins as inhibitors of aromatase, a potential therapeutic protein target for the treatment of breast cancer. Herein, a series of 3,7- and 4,7-disubstituted coumarin derivatives (1-34) with R1 and R2 substituents bearing aromatase inhibitory activity were modeled as a function of molecular and quantum chemical descriptors derived from low-energy conformer geometrically optimized at B3LYP/6-31G(d) level of theory. Insights on origins of aromatase inhibitory activity was afforded by the computed set of 7 descriptors comprising of F10[N-O], Inflammat-50, Psychotic-80, H-047, BELe1, B10[C-O] and MAXDP. Such significant descriptors were used for QSAR model construction and results indicated that model 4 afforded the best statistical performance. Good predictive performance were achieved as verified from the internal (comprising the training and the leave-one-out cross-validation (LOO-CV) sets) and external sets affording the following statistical parameters: R (2) Tr = 0.9576 and RMSETr = 0.0958 for the training set; Q (2) CV = 0.9239 and RMSECV = 0.1304 for the LOO-CV set as well as Q (2) Ext = 0.7268 and RMSEExt = 0.2927 for the external set. Significant descriptors showed correlation with functional substituents, particularly, R1 in governing high potency as aromatase inhibitor. Molecular docking calculations suggest that key residues interacting with the coumarins were predominantly lipophilic or non-polar while a few were polar and positively-charged. Findings illuminated herein serve as the impetus that can be used to rationally guide the design of new aromatase inhibitors.

  8. Second locus involved in human immunodeficiency virus type 1 resistance to protease inhibitors.

    PubMed Central

    Doyon, L; Croteau, G; Thibeault, D; Poulin, F; Pilote, L; Lamarre, D

    1996-01-01

    Protease inhibitors are potent antiviral agents against human immunodeficiency virus type 1. As with reverse transcriptase inhibitors, however, resistance to protease inhibitors can develop and is attributed to the appearance of mutations in the protease gene. With the substrate analog protease inhibitors BILA 1906 BS and BILA 2185 BS, 350- to 1,500-fold-resistant variants have been selected in vitro and were found not only to contain mutations in the protease gene but also to contain mutations in Gag precursor p1/p6 and/or NC (p7)/p1 cleavage sites. Mutations in cleavage sites give rise to better peptide substrates for the protease in vitro and to improved processing of p15 precursors in drug-resistant clones. Importantly, removal of cleavage site mutations in resistant clones leads to a decrease or even an absence of viral growth, confirming their role in viral fitness. Therefore, these second-locus mutations indicate that cleavage of p15 is a rate-limiting step in polyprotein processing in highly resistant viruses. The functional constraint of p15 processing also suggests that additional selective pressure could further compromise viral fitness and maintain the benefits of antiviral therapies. PMID:8648711

  9. Equine cytochrome P450 aromatase exhibits an estrogen 2-hydroxylase activity in vitro.

    PubMed

    Almadhidi, J; Moslemi, S; Drosdowsky, M A; Séralini, G E

    1996-09-01

    Aromatase (estrogen synthetase) is a steroidogenic enzyme complex which catalyzes the conversion of androgens to estrogens (termed aromatization). This enzyme was purified from adult equine testis to homogeneity by five chromatographic steps. The ability of purified and reconstituted equine aromatase to exhibit an estrogen 2-hydroxylase activity was tested and compared to testosterone aromatization. Enzymatic activities were assessed by tritiated water release from labelled estradiol and testosterone. Kinetic analysis of estradiol 2-hydroxylation showed an apparent K(m) of 23 microM and a V(max) of 18 nmol/min/mg, whereas the values for testosterone aromatization were a K(m) of 15.7 nM and a V(max) of 34.6 pmol/min/mg. A specific antiserum raised against purified testicular equine P450arom and known to inhibit aromatase activity [1] was also found to inhibit the estrogen hydroxylase activity of equine placental microsomes in a dose-dependent manner with an IC50 value of 15 microl serum: 0.5 ml incubate. The estrogen hydroxylase activity was inhibited in a dose-dependent manner by two classes of aromatase inhibitors, i.e. steroidal-- (4-hydroxyandrostenedione and 7alpha-([4-aminophenyl]thio)-androst-4-ene-3, 17-dione)--and non-steroidal--(fadrozole and miconazole). The IC50 values were approximately 300 and 890 nM for 4-hydroxyandrostenedione and 7alpha-([4-aminophenyl]thio)-androst-4-ene-3, 17-dione, and 92 and 285 nM, for fadrozole and miconazole, respectively. Furthermore, 4-hydroxyandrostenedione caused a time-dependent inactivation of estrogen hydroxylase activity. We conclude that equine aromatase is able to use estradiol as a substrate, and converts it to catechol estradiol in vitro, possibly using the active site of aromatization. This is the first demonstration that equine aromatase functions as an estrogen 2-hydroxylase, in addition to transforming androgens into estrogen.

  10. Bioengineering resistance to phytoene desaturase inhibitors in Arabidopsis thaliana

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Three natural somatic mutations at codon 304 of the phytoene desaturase gene (pds) of Hydrilla verticillata ( L. f. Royle) have been reported to provide resistance to the herbicide fluridone. We substituted the arginine 304 present in the wild-type H. verticillata phytoene desaturase (PDS) with all...

  11. Efficacy of ponatinib against ABL tyrosine kinase inhibitor-resistant leukemia cells

    SciTech Connect

    Okabe, Seiichi Tauchi, Tetsuzo; Tanaka, Yuko; Ohyashiki, Kazuma

    2013-06-07

    Highlights: •Efficacy of ponatinib against ABL tyrosine kinase inhibitor-resistant leukemia cells okabe et al. •Imatinib or nilotinib resistance was involved Src family kinase. •The BCR-ABL point mutation (E334V) was highly resistant to imatinib or nilotinib. •Ponatinib was a powerful strategy against imatinib or nilotinib resistant Ph-positive cells. -- Abstract: Because a substantial number of patients with chronic myeloid leukemia acquire resistance to ABL tyrosine kinase inhibitors (TKIs), their management remains a challenge. Ponatinib, also known as AP24534, is an oral multi-targeted TKI. Ponatinib is currently being investigated in a pivotal phase 2 clinical trial. In the present study, we analyzed the molecular and functional consequences of ponatinib against imatinib- or nilotinib-resistant (R) K562 and Ba/F3 cells. The proliferation of imatinib- or nilotinib-resistant K562 cells did not decrease after treatment with imatinib or nilotinib. Src family kinase Lyn was activated. Point mutation Ba/F3 cells (E334 V) were also highly resistant to imatinib and nilotinib. Treatment with ponatinib for 72 h inhibited the growth of imatinib- and nilotinib-resistant cells. The phosphorylation of BCR-ABL, Lyn, and Crk-L was reduced. This study demonstrates that ponatinib has an anti-leukemia effect by reducing ABL and Lyn kinase activity and this information may be of therapeutic relevance.

  12. Prolonged exposure to FLT3 inhibitors leads to resistance via activation of parallel signaling pathways

    PubMed Central

    Piloto, Obdulio; Wright, Melissa; Brown, Patrick; Kim, Kyu-Tae; Levis, Mark; Small, Donald

    2007-01-01

    Continuous treatment of malignancies with tyrosine kinase inhibitors (TKIs) may select for resistant clones (ie, imatinib mesylate). To study resistance to TKIs targeting FLT3, a receptor tyrosine kinase that is frequently mutated in acute myelogenous leukemia (AML), we developed resistant human cell lines through prolonged coculture with FLT3 TKIs. FLT3 TKI-resistant cell lines and primary samples still exhibit inhibition of FLT3 phosphorylation on FLT3 TKI treatment. However, FLT3 TKI-resistant cell lines and primary samples often show continued activation of downstream PI3K/Akt and/or Ras/MEK/MAPK signaling pathways as well as continued expression of genes involved in FLT3-mediated cellular transformation. Inhibition of these signaling pathways restores partial sensitivity to FLT3 TKIs. Mutational screening of FLT3 TKI-resistant cell lines revealed activating N-Ras mutations in 2 cell lines that were not present in the parental FLT3 TKI-sensitive cell line. Taken together, these data indicate that FLT3 TKI-resistant cells most frequently become FLT3 independent because of activation of parallel signaling pathways that provide compensatory survival/proliferation signals when FLT3 is inhibited. Anti-FLT3 mAb treatment was still cytotoxic to FLT3 TKI-resistant clones. An approach combining FLT3 TKIs with anti-FLT3 antibodies and/or inhibitors of important pathways downstream of FLT3 may reduce the chances of developing resistance. PMID:17047150

  13. Effects of AKT inhibitor therapy in response and resistance to BRAF inhibition in melanoma

    PubMed Central

    2014-01-01

    Background The clinical use of BRAF inhibitors for treatment of metastatic melanoma is limited by the development of drug resistance. In this study we investigated whether co-targeting the MAPK and the PI3K-AKT pathway can prevent emergence of resistance or provide additional growth inhibitory effects in vitro. Methods Anti-tumor effects of the combination of the BRAF inhibitor (BRAFi) dabrafenib and GSK2141795B (AKTi) in a panel of 23 BRAF mutated melanoma cell lines were evaluated on growth inhibition by an ATP-based luminescent assay, on cell cycle and apoptosis by flow cytometry and on cell signaling by western blot. Moreover, we investigated the possibilities of delaying or reversing resistance or achieving further growth inhibition by combining AKTi with dabrafenib and/or the MEK inhibitor (MEKi) trametinib by using long term cultures. Results More than 40% of the cell lines, including PTEN-/- and AKT mutants showed sensitivity to AKTi (IC50 < 1.5 μM). The combination of dabrafenib and AKTi synergistically potentiated growth inhibition in the majority of cell lines with IC50 > 5 nM dabrafenib. Combinatorial treatment induced apoptosis only in cell lines sensitive to AKTi. In long term cultures of a PTEN-/- cell line, combinatorial treatment with the MAPK inhibitors, dabrafenib and trametinib, and AKTi markedly delayed the emergence of drug resistance. Moreover, combining AKTi with the MAPK inhibitors from the beginning provided superior growth inhibitory effects compared to addition of AKTi upon development of resistance to MAPK inhibitors in this particular cell line. Conclusions AKTi combined with BRAFi-based therapy may benefit patients with tumors harboring BRAF mutations and particularly PTEN deletions or AKT mutations. PMID:24735930

  14. Exocytosis of polyubiquitinated proteins in bortezomib-resistant leukemia cells: a role for MARCKS in acquired resistance to proteasome inhibitors

    PubMed Central

    Franke, Niels E.; Kaspers, Gertjan L.; Assaraf, Yehuda G.; van Meerloo, Johan; Niewerth, Denise; Kessler, Floortje L.; Poddighe, Pino J.; Kole, Jeroen; Smeets, Serge J.; Ylstra, Bauke; Bi, Chonglei; Chng, Wee Joo; Horton, Terzah M.; Menezes, Rene X.; Musters, Renée J.P.; Zweegman, Sonja; Jansen, Gerrit; Cloos, Jacqueline

    2016-01-01

    PSMB5 mutations and upregulation of the β5 subunit of the proteasome represent key determinants of acquired resistance to the proteasome inhibitor bortezomib (BTZ) in leukemic cells in vitro. We here undertook a multi-modality (DNA, mRNA, miRNA) array-based analysis of human CCRF-CEM leukemia cells and BTZ-resistant subclones to determine whether or not complementary mechanisms contribute to BTZ resistance. These studies revealed signatures of markedly reduced expression of proteolytic stress related genes in drug resistant cells over a broad range of BTZ concentrations along with a high upregulation of myristoylated alanine-rich C-kinase substrate (MARCKS) gene expression. MARCKS upregulation was confirmed on protein level and also observed in other BTZ-resistant tumor cell lines as well as in leukemia cells with acquired resistance to other proteasome inhibitors. Moreover, when MARCKS protein expression was demonstrated in specimens derived from therapy-refractory pediatric leukemia patients (n = 44), higher MARCKS protein expression trended (p = 0.073) towards a dismal response to BTZ-containing chemotherapy. Mechanistically, we show a BTZ concentration-dependent association of MARCKS protein levels with the emergence of ubiquitin-containing vesicles in BTZ-resistant CEM cells. These vesicles were found to be extruded and taken up in co-cultures with proteasome-proficient acceptor cells. Consistent with these observations, MARCKS protein associated with ubiquitin-containing vesicles was also more prominent in clinical leukemic specimen with ex vivo BTZ resistance compared to BTZ-sensitive leukemia cells. Collectively, we propose a role for MARCKS in a novel mechanism of BTZ resistance via exocytosis of ubiquitinated proteins in BTZ-resistant cells leading to quenching of proteolytic stress. PMID:27542283

  15. Aromatase Inhibition Reduces Insulin Sensitivity in Healthy Men

    PubMed Central

    Homer, Natalie Z. M.; Faqehi, Abdullah M. M.; Upreti, Rita; Livingstone, Dawn E.; McInnes, Kerry J.; Andrew, Ruth; Walker, Brian R.

    2016-01-01

    Context: Deficiency of aromatase, the enzyme that catalyzes the conversion of androgens to estrogens, is associated with insulin resistance in humans and mice. Objective: We hypothesized that pharmacological aromatase inhibition results in peripheral insulin resistance in humans. Design: This was a double-blind, randomized, controlled, crossover study. Setting: The study was conducted at a clinical research facility. Participants: Seventeen healthy male volunteers (18–50 y) participated in the study. Intervention: The intervention included oral anastrozole (1 mg daily) and placebo, each for 6 weeks with a 2-week washout period. Main Outcome Measure: Glucose disposal and rates of lipolysis were measured during a stepwise hyperinsulinemic euglycemic clamp. Data are mean (SEM). Results: Anastrozole therapy resulted in significant estradiol suppression (59.9 ± 3.6 vs 102.0 ± 5.7 pmol/L, P = < .001) and a more modest elevation of total T (25.8 ± 1.2 vs 21.4 ± 0.7 nmol/L, P = .003). Glucose infusion rate, during the low-dose insulin infusion, was lower after anastrozole administration (12.16 ± 1.33 vs 14.15 ± 1.55 μmol/kg·min, P = .024). No differences in hepatic glucose production or rate of lipolysis were observed. Conclusion: Aromatase inhibition reduces insulin sensitivity, with respect to peripheral glucose disposal, in healthy men. Local generation and action of estradiol, at the level of skeletal muscle, is likely to be an important determinant of insulin sensitivity. PMID:26967690

  16. Hope and Disappointment: Covalent Inhibitors to Overcome Drug Resistance in Non-Small Cell Lung Cancer

    PubMed Central

    2015-01-01

    In the last five years, the detailed understanding of how to overcome T790M drug resistance in non-small cell lung cancer (NSCLC) has culminated in the development of a third-generation of covalent EGFR inhibitors with excellent clinical outcomes. However, the emergence of a newly discovered acquired drug resistance challenges the concept of small molecule targeted cancer therapy in NSCLC. PMID:26819655

  17. Viral resistance to human immunodeficiency virus type 1-specific pyridinone reverse transcriptase inhibitors.

    PubMed Central

    Nunberg, J H; Schleif, W A; Boots, E J; O'Brien, J A; Quintero, J C; Hoffman, J M; Emini, E A; Goldman, M E

    1991-01-01

    Human immunodeficiency virus type 1 (HIV-1)-specific pyridinone reverse transcriptase (RT) inhibitors prevent HIV-1 replication in cell culture (M. E. Goldman, J. H. Nunberg, J. A. O'Brien, J.C. Quintero, W. A. Schleif, K. F. Freund, S. L. Gaul, W. S. Saari, J. S. Wai, J. M. Hoffman, P. S. Anderson, D. J. Hupe, E. A. Emini, and A. M. Stern, Proc. Natl. Acad. Sci. USA 88:6863-6867, 1991). In contrast to nucleoside analog inhibitors, such as AZT, which need to be converted to triphosphates by host cells, these compounds act directly to inhibit RT via a mechanism which is noncompetitive with respect to deoxynucleoside triphosphates. As one approach to define the mechanism of action of pyridinone inhibitors, we isolated resistant mutants of HIV-1 in cell culture. Serial passage in the presence of inhibitor yielded virus which was 1,000-fold resistant to compounds of this class. Bacterially expressed RTs molecularly cloned from resistant viruses were also resistant. The resistant RT genes encoded two amino acid changes, K-103 to N and Y-181 to C, each of which contributed partial resistance. The mutation at amino acid 181 lies adjacent to the conserved YG/MDD motif found in most DNA and RNA polymerases. The mutation at amino acid 103 lies within a region of RT which may be involved in PPi binding. The resistant viruses, although sensitive to nucleoside analogs, were cross-resistant to the structurally unrelated RT inhibitors TIBO R82150 (R. Pauwels, K. Andries, J. Desmyter, D. Schols, M. J. Kukla, H. J. Breslin, A. Raeymaeckers, J. Van Gelder, R. Woestenborghs, J. Heykanti, K. Schellekens, M. A. C. Janssen, E. De Clercq, and P. A. J. Janssen, Nature [London] 343:470-474, 1990) and BI-RG-587 (V. J. Merluzzi, K. D. Hargrave, M. Labadia, K. Grozinger, M. Skoog, J. C. Wu, C.-K. Shih, K. Eckner, S. Hattox, J. Adams, A. S. Rosenthal, R. Faanes, R. J. Eckner, R. A. Koup, and J. L. Sullivan, Science 250:1411-1413, 1990). Thus, these nonnucleoside analog inhibitors may share a

  18. Photosensitive dissolution inhibitors and resists based on onium salt carboxylates

    DOEpatents

    Dentinger, Paul M.; Simison, Kelby L.

    2005-11-29

    A photoresist composition that employs onium salt carboxylates as thermally stable dissolution inhibitors. The photoresist composition can be either an onium carboxylate salt with a phenolic photoresist, such as novolac, or an onium cation protected carboxylate-containing resin such as an acrylic/acrylic acid copolymer. The onium carboxylate can be an onium cholate, wherein the onium cholate is an iodonium cholate. Particularly preferred iodonium cholates are alkyloxyphenylphenyl iodonium cholates and most particularly preferred is octyloxyphenyphenyl iodonium cholate. The photoresist composition will not create nitrogen or other gaseous byproducts upon exposure to radiation, does not require water for photoactivation, has acceptable UV radiation transmission characteristics, and is thermally stable at temperatures required for solvent removal.

  19. RecA Inhibitors Potentiate Antibiotic Activity and Block Evolution of Antibiotic Resistance.

    PubMed

    Alam, Md Kausar; Alhhazmi, Areej; DeCoteau, John F; Luo, Yu; Geyer, C Ronald

    2016-03-17

    Antibiotic resistance arises from the maintenance of resistance mutations or genes acquired from the acquisition of adaptive de novo mutations or the transfer of resistance genes. Antibiotic resistance is acquired in response to antibiotic therapy by activating SOS-mediated DNA repair and mutagenesis and horizontal gene transfer pathways. Initiation of the SOS pathway promotes activation of RecA, inactivation of LexA repressor, and induction of SOS genes. Here, we have identified and characterized phthalocyanine tetrasulfonic acid RecA inhibitors that block antibiotic-induced activation of the SOS response. These inhibitors potentiate the activity of bactericidal antibiotics, including members of the quinolone, β-lactam, and aminoglycoside families in both Gram-negative and Gram-positive bacteria. They reduce the ability of bacteria to acquire antibiotic resistance mutations and to transfer mobile genetic elements conferring resistance. This study highlights the advantage of including RecA inhibitors in bactericidal antibiotic therapies and provides a new strategy for prolonging antibiotic shelf life.

  20. Circulating tumour DNA profiling reveals heterogeneity of EGFR inhibitor resistance mechanisms in lung cancer patients.

    PubMed

    Chabon, Jacob J; Simmons, Andrew D; Lovejoy, Alexander F; Esfahani, Mohammad S; Newman, Aaron M; Haringsma, Henry J; Kurtz, David M; Stehr, Henning; Scherer, Florian; Karlovich, Chris A; Harding, Thomas C; Durkin, Kathleen A; Otterson, Gregory A; Purcell, W Thomas; Camidge, D Ross; Goldman, Jonathan W; Sequist, Lecia V; Piotrowska, Zofia; Wakelee, Heather A; Neal, Joel W; Alizadeh, Ash A; Diehn, Maximilian

    2016-06-10

    Circulating tumour DNA (ctDNA) analysis facilitates studies of tumour heterogeneity. Here we employ CAPP-Seq ctDNA analysis to study resistance mechanisms in 43 non-small cell lung cancer (NSCLC) patients treated with the third-generation epidermal growth factor receptor (EGFR) inhibitor rociletinib. We observe multiple resistance mechanisms in 46% of patients after treatment with first-line inhibitors, indicating frequent intra-patient heterogeneity. Rociletinib resistance recurrently involves MET, EGFR, PIK3CA, ERRB2, KRAS and RB1. We describe a novel EGFR L798I mutation and find that EGFR C797S, which arises in ∼33% of patients after osimertinib treatment, occurs in <3% after rociletinib. Increased MET copy number is the most frequent rociletinib resistance mechanism in this cohort and patients with multiple pre-existing mechanisms (T790M and MET) experience inferior responses. Similarly, rociletinib-resistant xenografts develop MET amplification that can be overcome with the MET inhibitor crizotinib. These results underscore the importance of tumour heterogeneity in NSCLC and the utility of ctDNA-based resistance mechanism assessment.

  1. Circulating tumour DNA profiling reveals heterogeneity of EGFR inhibitor resistance mechanisms in lung cancer patients

    PubMed Central

    Chabon, Jacob J.; Simmons, Andrew D.; Lovejoy, Alexander F.; Esfahani, Mohammad S.; Newman, Aaron M.; Haringsma, Henry J.; Kurtz, David M.; Stehr, Henning; Scherer, Florian; Karlovich, Chris A.; Harding, Thomas C.; Durkin, Kathleen A.; Otterson, Gregory A.; Purcell, W. Thomas; Camidge, D. Ross; Goldman, Jonathan W.; Sequist, Lecia V.; Piotrowska, Zofia; Wakelee, Heather A.; Neal, Joel W.; Alizadeh, Ash A.; Diehn, Maximilian

    2016-01-01

    Circulating tumour DNA (ctDNA) analysis facilitates studies of tumour heterogeneity. Here we employ CAPP-Seq ctDNA analysis to study resistance mechanisms in 43 non-small cell lung cancer (NSCLC) patients treated with the third-generation epidermal growth factor receptor (EGFR) inhibitor rociletinib. We observe multiple resistance mechanisms in 46% of patients after treatment with first-line inhibitors, indicating frequent intra-patient heterogeneity. Rociletinib resistance recurrently involves MET, EGFR, PIK3CA, ERRB2, KRAS and RB1. We describe a novel EGFR L798I mutation and find that EGFR C797S, which arises in ∼33% of patients after osimertinib treatment, occurs in <3% after rociletinib. Increased MET copy number is the most frequent rociletinib resistance mechanism in this cohort and patients with multiple pre-existing mechanisms (T790M and MET) experience inferior responses. Similarly, rociletinib-resistant xenografts develop MET amplification that can be overcome with the MET inhibitor crizotinib. These results underscore the importance of tumour heterogeneity in NSCLC and the utility of ctDNA-based resistance mechanism assessment. PMID:27283993

  2. Two natural products, trans-phytol and (22E)-ergosta-6,9,22-triene-3β,5α,8α-triol, inhibit the biosynthesis of estrogen in human ovarian granulosa cells by aromatase (CYP19).

    PubMed

    Guo, Jiajia; Yuan, Yun; Lu, Danfeng; Du, Baowen; Xiong, Liang; Shi, Jiangong; Yang, Lijuan; Liu, Wanli; Yuan, Xiaohong; Zhang, Guolin; Wang, Fei

    2014-08-15

    Aromatase is the only enzyme in vertebrates to catalyze the biosynthesis of estrogens. Although inhibitors of aromatase have been developed for the treatment of estrogen-dependent breast cancer, the whole-body inhibition of aromatase causes severe adverse effects. Thus, tissue-selective aromatase inhibitors are important for the treatment of estrogen-dependent cancers. In this study, 63 natural products with diverse structures were examined for their effects on estrogen biosynthesis in human ovarian granulosa-like KGN cells. Two compounds-trans-phytol (SA-20) and (22E)-ergosta-6,9,22-triene-3β,5α,8α-triol (SA-48)-were found to potently inhibit estrogen biosynthesis (IC50: 1μM and 0.5μM, respectively). Both compounds decreased aromatase mRNA and protein expression levels in KGN cells, but had no effect on the aromatase catalytic activity in aromatase-overexpressing HEK293A cells and recombinant expressed aromatase. The two compounds decreased the expression of aromatase promoter I.3/II. Neither compound affected intracellular cyclic AMP (cAMP) levels, but they inhibited the phosphorylation or protein expression of cAMP response element-binding protein (CREB). The effects of these two compounds on extracellular regulated kinase (ERK), c-Jun N-terminal kinase (JNK), p38 mitogen-activated protein kinases (MAPKs), and AKT/phosphoinositide 3-kinase (PI3K) pathway were examined. Inhibition of p38 MAPK could be the mechanism underpinning the actions of these compounds. Our results suggests that natural products structurally similar to SA-20 and SA-48 may be a new source of tissue-selective aromatase modulators, and that p38 MAPK is important in the basal control of aromatase in ovarian granulosa cells. SA-20 and SA-48 warrant further investigation as new pharmaceutical tools for the prevention and treatment of estrogen-dependent cancers.

  3. Investigating the Regulation and Potential Role of Nonhypoxic Hypoxia-Inducible Factor 1 (HIF-1) in Aromatase Inhibitor Resistant Breast Cancer

    DTIC Science & Technology

    2013-10-01

    hypoxia conditions (1% O2). Total mRNA was extracted and vimentin and 18S rRNA were analyzed by real-time RT- PCR . Real - time results are expressed as...endothelin 1, and fibronectin mRNA, and 18S rRNA were analyzed by real-time RT- PCR . Real - time results are expressed as the fold-change in mRNA levels

  4. Investigation the Regulation and Potential Role of Nonhypoic Hypoxia-Inducible Factor 1 (HIF-1) in Aromatase Inhibitor-Resistant Breast Cancer

    DTIC Science & Technology

    2014-10-01

    another transcription factor. Both TWIST (Sun et al. 2009) and Snail (Zhang et al. 2013) transcription factors are known to be direct HIF-1 target...genes. In turn, both Snail (Kaufhold et al. 2014) and Twist (Yang et al. 2004) are known to regulate vimentin expression.. 4 Although TWIST and... Snail were not upregulated LTLTCa vs. MCF-7Ca cells (refer to 2013 annual summary), their functions as transcription factors may be upregulated in

  5. Characterization of Inhibitor-Resistant TEM β-Lactamases and Mechanisms of Fluoroquinolone Resistance in Escherichia coli Isolates.

    PubMed

    Ríos, Esther; López, Maria Carmen; Rodríguez-Avial, Iciar; Pena, Irene; Picazo, Juan Jose

    2015-10-01

    The aim of present work was to characterize the inhibitor-resistant TEM (IRT) β-lactamases produced by Escherichia coli in Hospital Clínico San Carlos (Madrid, Spain). Mechanisms of fluoroquinolone resistance among IRT-producing strains were also studied. Isolates with susceptibility to cephalosporins and amoxicillin-clavulanate (AMC) resistance were collected in our hospital (November 2011-July 2012) from both outpatients and hospitalized patients. Among 70 AMC-resistant E. coli strains, 28 (40%) produced IRT enzymes. Most of them were uropathogens (82.1%) and recovered from outpatients (75%). Seven different IRT enzymes were identified with TEM-30 (IRT-2) being the most prevalent, followed by TEM-40 (IRT-11). A high rate of ciprofloxacin resistance was found among IRT-producing strains (50%). Most of the ciprofloxacin-resistant isolates showed ciprofloxacin minimum inhibitory concentration >32 mg/L and contained two mutations in both gyrA and parC genes. Four IRT enzyme producers harbored the qnr gene. ST131 clone was mainly responsible for both IRT enzyme production and ciprofloxacin resistance. In conclusion, data from this study show that the frequency of IRT producers was 40% and a high rate of ciprofloxacin resistance was found among IRT-producing isolates. Current and future actions should be taken into account to avoid or reduce the development of AMC and fluoroquinolone resistance in E. coli.

  6. Insect resistance to sugar beet pests mediated by a Beta vulgaris proteinase inhibitor transgene

    Technology Transfer Automated Retrieval System (TEKTRAN)

    We transformed sugar beet (Beta vulgaris) hairy roots and Nicotiana benthamiana plants with a Beta vulgaris root gene (BvSTI) that codes for a serine proteinase inhibitor. BvSTI is a root gene cloned from the F1016 breeding line that has moderate levels of resistance to the sugar beet root maggot ...

  7. Mammographic Breast Density Response to Aromatase Inhibition

    PubMed Central

    Vachon, Celine M.; Suman, Vera J.; Brandt, Kathleen R.; Kosel, Matthew L.; Buzdar, Aman U.; Olson, Janet E.; Wu, Fang-Fang; Flickinger, Lynn M.; Ursin, Giske; Elliott, Catherine R.; Shepherd, Lois; Weinshilboum, Richard M.; Goss, Paul E.; Ingle, James N.

    2013-01-01

    Purpose Mammographic breast density (MBD) is decreased by tamoxifen, but the effect of aromatase inhibitors (AI) is less clear. Experimental Design We enrolled early stage postmenopausal breast cancer patients initiating adjuvant AI therapy and ascertained mammograms before and at an average 10 months of AI therapy. We matched cases to healthy postmenopausal women (controls) from a large mammography screening cohort on age, baseline body mass index, baseline MBD and interval between mammograms. We estimated change in MBD using a computer-assisted thresholding program (Cumulus) and compared differences between cases and matched controls. Results In predominantly white women (96%), we found 14% of the 387 eligible cases had a MBD reduction of at least 5% after an average of 10 months of AI therapy. MBD reductions were associated with higher baseline MBD, AI use for more than 12 months and prior postmenopausal hormone use. Comparing each case to her matched control, there was no evidence of an association of change in MBD with AI therapy (median case-control difference among 369 pairs was −0.1% (10th and 90th percentile: −5.9%, 5.2%) p=0.51). Case-control differences were similar by type of AI (p’s 0.41 and 0.56); prior use of postmenopausal hormones (p=0.85); baseline MBD (p=0.55); or length of AI therapy (p=0.08). Conclusions In postmenopausal women treated with AIs, 14% of cases had a MBD reduction of >5%, but these decreases did not differ from matched controls. These data suggest that MBD is not a clinically useful biomarker for predicting the value of AI therapy in white postmenopausal women. PMID:23468058

  8. Use of resistant ACCase mutants to screen for novel inhibitors against resistant and susceptible forms of ACCase from grass weeds.

    PubMed

    Shukla, Amit; Nycholat, Corwin; Subramanian, Mani V; Anderson, Richard J; Devine, Malcolm D

    2004-08-11

    The aryloxyphenoxypropionic acid (AOPP) and cyclohexanedione (CHD) herbicides inhibit the first committed enzyme in fatty acid biosynthesis, acetyl CoA carboxylase (ACCase). The frequent use of AOPP and CHD herbicides has resulted in the development of resistance to these herbicides in many grass weed species. New herbicides that inhibit both the susceptible and resistant forms of ACCase in grass weeds would have obvious commercial appeal. In the present study, an attempt was made to identify molecules that target both the herbicide-sensitive and -resistant forms of ACCase. Seven experimental compounds, either CHD-like or AOPP-CHD hybrids, were synthesized and assayed against previously characterized susceptible and resistant forms of ACCase. All seven compounds inhibited ACCase from sensitive biotypes of Setaria viridis and Eleusine indica (I50 values from 6.4 to >100 microM) but were not particularly potent compared to some commercialized herbicides (I50 values of 0.08-5.6 microM). In almost all cases, the I50 values for each compound assayed against the resistant ACCases were higher than those against the corresponding sensitive ACCase, indicating reduced binding to the resistant ACCases. One compound, a CHD analogue, was almost equally effective against the resistant and susceptible ACCases, although it was not a very potent ACCase inhibitor per se (I50 of 51 and 76 microM against susceptible ACCase from S. viridis and E. indica, respectively). The AOPP-CHD hybrid molecules also inhibited some of the resistant ACCases, with I50 values ranging from 6.4 to 50 microM. These compounds may be good leads for developing ACCase inhibitors that target a wider range of ACCase isoforms, including those found in AOPP- and CHD-resistant weed biotypes.

  9. Emergence of H7N9 Influenza A Virus Resistant to Neuraminidase Inhibitors in Nonhuman Primates

    PubMed Central

    Shichinohe, Shintaro; Nakayama, Misako; Igarashi, Manabu; Ishii, Akihiro; Ishigaki, Hirohito; Ishida, Hideaki; Kitagawa, Naoko; Sasamura, Takako; Shiohara, Masanori; Doi, Michiko; Tsuchiya, Hideaki; Nakamura, Shinichiro; Okamatsu, Masatoshi; Sakoda, Yoshihiro; Kida, Hiroshi

    2015-01-01

    The number of patients infected with H7N9 influenza virus has been increasing since 2013. We examined the efficacy of neuraminidase (NA) inhibitors and the efficacy of a vaccine against an H7N9 influenza virus, A/Anhui/1/2013 (H7N9), isolated from a patient in a cynomolgus macaque model. NA inhibitors (oseltamivir and peramivir) barely reduced the total virus amount because of the emergence of resistant variants with R289K or I219T in NA [residues 289 and 219 in N9 of A/Anhui/1/2013 (H7N9) correspond to 292 and 222 in N2, respectively] in three of the six treated macaques, whereas subcutaneous immunization of an inactivated vaccine derived from A/duck/Mongolia/119/2008 (H7N9) prevented propagation of A/Anhui/1/2013 (H7N9) in all vaccinated macaques. The percentage of macaques in which variant H7N9 viruses with low sensitivity to the NA inhibitors were detected was much higher than that of macaques in which variant H5N1 highly pathogenic influenza virus was detected after treatment with one of the NA inhibitors in our previous study. The virus with R289K in NA was reported in samples from human patients, whereas that with I219T in NA was identified for the first time in this study using macaques, though no variant H7N9 virus was reported in previous studies using mice. Therefore, the macaque model enables prediction of the frequency of emerging H7N9 virus resistant to NA inhibitors in vivo. Since H7N9 strains resistant to NA inhibitors might easily emerge compared to other influenza viruses, monitoring of the emergence of variants is required during treatment of H7N9 influenza virus infection with NA inhibitors. PMID:26055368

  10. Carfilzomib and oprozomib synergize with histone deacetylase inhibitors in head and neck squamous cell carcinoma models of acquired resistance to proteasome inhibitors.

    PubMed

    Zang, Yan; Kirk, Christopher J; Johnson, Daniel E

    2014-09-01

    Acquired resistance to proteasome inhibitors represents a considerable impediment to their effective clinical application. Carfilzomib and its orally bioavailable structural analog oprozomib are second-generation, highly-selective, proteasome inhibitors. However, the mechanisms of acquired resistance to carfilzomib and oprozomib are incompletely understood, and effective strategies for overcoming this resistance are needed. Here, we developed models of acquired resistance to carfilzomib in two head and neck squamous cell carcinoma cell lines, UMSCC-1 and Cal33, through gradual exposure to increasing drug concentrations. The resistant lines R-UMSCC-1 and R-Cal33 demonstrated 205- and 64-fold resistance, respectively, relative to the parental lines. Similarly, a high level of cross-resistance to oprozomib, as well as paclitaxel, was observed, whereas only moderate resistance to bortezomib (8- to 29-fold), and low level resistance to cisplatin (1.5- to 5-fold) was seen. Synergistic induction of apoptosis signaling and cell death, and inhibition of colony formation followed co-treatment of acquired resistance models with carfilzomib and the histone deacetylase inhibitor (HDACi) vorinostat. Synergism was also seen with other combinations, including oprozomib plus vorinostat, or carfilzomib plus the HDACi entinostat. Synergism was accompanied by upregulation of proapoptotic Bik, and suppression of Bik attenuated the synergy. The acquired resistance models also exhibited elevated levels of MDR-1/P-gp. Inhibition of MDR-1/P-gp with reversin 121 partially overcame carfilzomib resistance in R-UMSCC-1 and R-Cal33 cells. Collectively, these studies indicate that combining carfilzomib or oprozomib with HDAC or MDR-1/P-gp inhibitors may be a useful strategy for overcoming acquired resistance to these proteasome inhibitors.

  11. Trypsin inhibitors from Capsicum baccatum var. pendulum leaves involved in Pepper yellow mosaic virus resistance.

    PubMed

    Moulin, M M; Rodrigues, R; Ribeiro, S F F; Gonçalves, L S A; Bento, C S; Sudré, C P; Vasconcelos, I M; Gomes, V M

    2014-11-07

    Several plant organs contain proteinase inhibitors, which are produced during normal plant development or are induced upon pathogen attack to suppress the enzymatic activity of phytopathogenic microorganisms. In this study, we examined the presence of proteinase inhibitors, specifically trypsin inhibitors, in the leaf extract of Capsicum baccatum var. pendulum inoculated with PepYMV (Pepper yellow mosaic virus). Leaf extract from plants with the accession number UENF 1624, which is resistant to PepYMV, was collected at 7 different times (0, 24, 48, 72, 96, 120, and 144 h). Seedlings inoculated with PepYMV and control seedlings were grown in a growth chamber. Protein extract from leaf samples was partially purified by reversed-phase chromatography using a C2/C18 column. Residual trypsin activity was assayed to detect inhibitors followed by Tricine-SDS-PAGE analysis to determine the N-terminal peptide sequence. Based on trypsin inhibitor assays, trypsin inhibitors are likely constitutively synthesized in C. baccatum var. pendulum leaf tissue. These inhibitors are likely a defense mechanism for the C. baccatum var. pendulum- PepYMV pathosystem.

  12. Structural insight into selectivity and resistance profiles of ROS1 tyrosine kinase inhibitors

    PubMed Central

    Davare, Monika A.; Vellore, Nadeem A.; Wagner, Jacob P.; Eide, Christopher A.; Goodman, James R.; Drilon, Alexander; Deininger, Michael W.; O’Hare, Thomas; Druker, Brian J.

    2015-01-01

    Oncogenic ROS1 fusion proteins are molecular drivers in multiple malignancies, including a subset of non-small cell lung cancer (NSCLC). The phylogenetic proximity of the ROS1 and anaplastic lymphoma kinase (ALK) catalytic domains led to the clinical repurposing of the Food and Drug Administration (FDA)-approved ALK inhibitor crizotinib as a ROS1 inhibitor. Despite the antitumor activity of crizotinib observed in both ROS1- and ALK-rearranged NSCLC patients, resistance due to acquisition of ROS1 or ALK kinase domain mutations has been observed clinically, spurring the development of second-generation inhibitors. Here, we profile the sensitivity and selectivity of seven ROS1 and/or ALK inhibitors at various levels of clinical development. In contrast to crizotinib’s dual ROS1/ALK activity, cabozantinib (XL-184) and its structural analog foretinib (XL-880) demonstrate a striking selectivity for ROS1 over ALK. Molecular dynamics simulation studies reveal structural features that distinguish the ROS1 and ALK kinase domains and contribute to differences in binding site and kinase selectivity of the inhibitors tested. Cell-based resistance profiling studies demonstrate that the ROS1-selective inhibitors retain efficacy against the recently reported CD74-ROS1G2032R mutant whereas the dual ROS1/ALK inhibitors are ineffective. Taken together, inhibitor profiling and stringent characterization of the structure–function differences between the ROS1 and ALK kinase domains will facilitate future rational drug design for ROS1- and ALK-driven NSCLC and other malignancies. PMID:26372962

  13. Pharmacological concentration of resveratrol suppresses aromatase in JEG-3 cells.

    PubMed

    Wang, Yun; Leung, Lai K

    2007-09-28

    Estrogen is crucial in preparing of pregnancy, and its role in the maintenance of pregnancy has yet to be elucidated. During the course of pregnancy, the placenta is responsible for the provision of estrogen. The hormone biosynthesis is catalyzed by cytochrome P450 (CYP) 19 or aromatase. In the present study, we screened several common dietary components and identified the grape polyphenol resveratrol to be a potential inhibitor in the hormone synthesis. In a recombinant protein system resveratrol inhibited the aromatase activity with an IC(50) value of approximately 40 microM. Subsequent analysis was performed in the human placental JEG-3 cells, and 25 microM resveratrol significantly reduced the mRNA abundance in these cells. Since the transcriptional control of CYP19 gene is tissue-specific and the proximal promoter region of exon Ia has previously been shown to be crucial in CYP19 expression in placental cells, we also evaluated the promoter activity of this gene. Reporter gene assays revealed that resveratrol repressed the transcriptional control of promoter Ia. The present study illustrated the possibility that dietary supplementation of resveratrol interfered with the normal functioning of placental cells.

  14. Defining the Biological Domain of Applicability of Adverse Outcome Pathways Across Diverse Species: The Estrogen Receptor/Aromatase Case Study

    EPA Science Inventory

    Aromatase inhibitors (e.g. fadrozole, prochloraz) and estrogen receptor antagonists (e.g. tamoxifen) reduce the circulating concentration of 17β-estradiol, leading to reproductive dysfunction in affected organisms. While these toxic effects are well-characterized in fish and...

  15. Widespread potential for growth-factor-driven resistance to anticancer kinase inhibitors

    PubMed Central

    Wilson, Timothy R.; Fridlyand, Jane; Yan, Yibing; Penuel, Elicia; Burton, Luciana; Chan, Emily; Peng, Jing; Lin, Eva; Wang, Yulei; Sosman, Jeff; Ribas, Antoni; Li, Jiang; Moffat, John; Sutherlin, Daniel P.; Koeppen, Hartmut; Merchant, Mark; Neve, Richard; Settleman, Jeff

    2013-01-01

    Mutationally activated kinases define a clinically validated class of targets for cancer drug therapy1. However, the efficacy of kinase inhibitors in patients whose tumours harbour such alleles is invariably limited by innate or acquired drug resistance2,3. The identification of resistance mechanisms has revealed a recurrent theme—the engagement of survival signals redundant to those transduced by the targeted kinase4. Cancer cells typically express multiple receptor tyrosine kinases (RTKs) that mediate signals that converge on common critical downstream cell-survival effectors—most notably, phosphatidylinositol-3-OH kinase (PI(3)K) and mitogen-activated protein kinase (MAPK)5. Consequently, an increase in RTK-ligand levels, through autocrine tumour-cell production, paracrine contribution from tumour stroma6 or systemic production, could confer resistance to inhibitors of an oncogenic kinase with a similar signalling output. Here, using a panel of kinase-‘addicted’ human cancer cell lines, we found that most cells can be rescued from drug sensitivity by simply exposing them to one or more RTK ligands. Among the findings with clinical implications was the observation that hepatocyte growth factor (HGF) confers resistance to the BRAF inhibitor PLX4032 (vemurafenib) in BRAF-mutant melanoma cells. These observations highlight the extensive redundancy of RTK-transduced signalling in cancer cells and the potentially broad role of widely expressed RTK ligands in innate and acquired resistance to drugs targeting oncogenic kinases. PMID:22763448

  16. Multicenter Quality Control of Hepatitis C Virus Protease Inhibitor Resistance Genotyping

    PubMed Central

    Larrat, Sylvie; Laperche, Syria; Le Guillou-Guillemette, Hélène; Legrand-Abravanel, Florence; Bouchardeau, Françoise; Pivert, Adeline; Henquell, Cécile; Mirand, Audrey; André-Garnier, Elisabeth; Giordanengo, Valérie; Lagathu, Gisèle; Thibault, Vincent; Scholtes, Caroline; Schvoerer, Evelyne; Gaudy-Graffin, Catherine; Maylin, Sarah; Trimoulet, Pascale; Brochot, Etienne; Hantz, Sébastien; Gozlan, Joël; Roque-Afonso, Anne-Marie; Soussan, Patrick; Plantier, Jean-Christophe; Charpentier, Charlotte; Chevaliez, Stéphane; Colson, Philippe; Mackiewicz, Vincent; Aguilera, Lina; Rosec, Sylvain; Gouriou, Stéphanie; Magnat, Nelly; Lunel-Fabiani, Françoise; Izopet, Jacques; Morand, Patrice; Payan, Christopher; Pawlotsky, Jean-Michel

    2013-01-01

    Hepatitis C virus (HCV) protease inhibitor resistance-associated substitutions are selected during triple-therapy breakthrough. This multicenter quality control study evaluated the expertise of 23 French laboratories in HCV protease inhibitor resistance genotyping. A panel of 12 well-defined blinded samples comprising two wild-type HCV strains, nine transcripts from synthetic NS3 mutant samples or from clinical strains, and one HCV RNA-negative sample was provided to the participating laboratories. The results showed that any laboratory with expertise in sequencing techniques should be able to provide reliable HCV protease inhibitor resistance genotyping. Only a 0.7% error rate was reported for the amino acid sites studied. The accuracy of substitution identification ranged from 75% to 100%, depending on the laboratory. Incorrect results were mainly related to the methodology used. The results could be improved by changing the primers and modifying the process in order to avoid cross-contamination. This study underlines the value of quality control programs for viral resistance genotyping, which is required prior to launching observational collaborative multicenter studies on HCV resistance to direct-acting antiviral agents. PMID:23426922

  17. Multicenter quality control of hepatitis C virus protease inhibitor resistance genotyping.

    PubMed

    Vallet, Sophie; Larrat, Sylvie; Laperche, Syria; Le Guillou-Guillemette, Hélène; Legrand-Abravanel, Florence; Bouchardeau, Françoise; Pivert, Adeline; Henquell, Cécile; Mirand, Audrey; André-Garnier, Elisabeth; Giordanengo, Valérie; Lagathu, Gisèle; Thibault, Vincent; Scholtes, Caroline; Schvoerer, Evelyne; Gaudy-Graffin, Catherine; Maylin, Sarah; Trimoulet, Pascale; Brochot, Etienne; Hantz, Sébastien; Gozlan, Joël; Roque-Afonso, Anne-Marie; Soussan, Patrick; Plantier, Jean-Christophe; Charpentier, Charlotte; Chevaliez, Stéphane; Colson, Philippe; Mackiewicz, Vincent; Aguilera, Lina; Rosec, Sylvain; Gouriou, Stéphanie; Magnat, Nelly; Lunel-Fabiani, Françoise; Izopet, Jacques; Morand, Patrice; Payan, Christopher; Pawlotsky, Jean-Michel

    2013-05-01

    Hepatitis C virus (HCV) protease inhibitor resistance-associated substitutions are selected during triple-therapy breakthrough. This multicenter quality control study evaluated the expertise of 23 French laboratories in HCV protease inhibitor resistance genotyping. A panel of 12 well-defined blinded samples comprising two wild-type HCV strains, nine transcripts from synthetic NS3 mutant samples or from clinical strains, and one HCV RNA-negative sample was provided to the participating laboratories. The results showed that any laboratory with expertise in sequencing techniques should be able to provide reliable HCV protease inhibitor resistance genotyping. Only a 0.7% error rate was reported for the amino acid sites studied. The accuracy of substitution identification ranged from 75% to 100%, depending on the laboratory. Incorrect results were mainly related to the methodology used. The results could be improved by changing the primers and modifying the process in order to avoid cross-contamination. This study underlines the value of quality control programs for viral resistance genotyping, which is required prior to launching observational collaborative multicenter studies on HCV resistance to direct-acting antiviral agents.

  18. A novel HDAC inhibitor, CG200745, inhibits pancreatic cancer cell growth and overcomes gemcitabine resistance

    PubMed Central

    Lee, Hee Seung; Park, Soo Been; Kim, Sun A; Kwon, Sool Ki; Cha, Hyunju; Lee, Do Young; Ro, Seonggu; Cho, Joong Myung; Song, Si Young

    2017-01-01

    Pancreatic cancer is predominantly lethal, and is primarily treated using gemcitabine, with increasing resistance. Therefore, novel agents that increase tumor sensitivity to gemcitabine are needed. Histone deacetylase (HDAC) inhibitors are emerging therapeutic agents, since HDAC plays an important role in cancer initiation and progression. We evaluated the antitumor effect of a novel HDAC inhibitor, CG200745, combined with gemcitabine/erlotinib on pancreatic cancer cells and gemcitabine-resistant pancreatic cancer cells. Three pancreatic cancer-cell lines were used to evaluate the antitumor effect of CG200745 combined with gemcitabine/erlotinib. CG200745 induced the expression of apoptotic proteins (PARP and caspase-3) and increased the levels of acetylated histone H3. CG200745 with gemcitabine/erlotinib showed significant growth inhibition and synergistic antitumor effects in vitro. In vivo, gemcitabine/erlotinib and CG200745 reduced tumor size up to 50%. CG200745 enhanced the sensitivity of gemcitabine-resistant pancreatic cancer cells to gemcitabine, and decreased the level of ATP-binding cassette-transporter genes, especially multidrug resistance protein 3 (MRP3) and MRP4. The novel HDAC inhibitor, CG200745, with gemcitabine/erlotinib had a synergistic anti-tumor effect on pancreatic cancer cells. CG200745 significantly improved pancreatic cancer sensitivity to gemcitabine, with a prominent antitumor effect on gemcitabine-resistant pancreatic cancer cells. Therefore, improved clinical outcome is expected in the future. PMID:28134290

  19. Perturbation biology nominates upstream-downstream drug combinations in RAF inhibitor resistant melanoma cells.

    PubMed

    Korkut, Anil; Wang, Weiqing; Demir, Emek; Aksoy, Bülent Arman; Jing, Xiaohong; Molinelli, Evan J; Babur, Özgün; Bemis, Debra L; Onur Sumer, Selcuk; Solit, David B; Pratilas, Christine A; Sander, Chris

    2015-08-18

    Resistance to targeted cancer therapies is an important clinical problem. The discovery of anti-resistance drug combinations is challenging as resistance can arise by diverse escape mechanisms. To address this challenge, we improved and applied the experimental-computational perturbation biology method. Using statistical inference, we build network models from high-throughput measurements of molecular and phenotypic responses to combinatorial targeted perturbations. The models are computationally executed to predict the effects of thousands of untested perturbations. In RAF-inhibitor resistant melanoma cells, we measured 143 proteomic/phenotypic entities under 89 perturbation conditions and predicted c-Myc as an effective therapeutic co-target with BRAF or MEK. Experiments using the BET bromodomain inhibitor JQ1 affecting the level of c-Myc protein and protein kinase inhibitors targeting the ERK pathway confirmed the prediction. In conclusion, we propose an anti-cancer strategy of co-targeting a specific upstream alteration and a general downstream point of vulnerability to prevent or overcome resistance to targeted drugs.

  20. A delayed, gonadotropin-dependent and growth-factor mediated activation of the ERK1/2 cascade negatively regulates aromatase expression in granulosa cells*

    PubMed Central

    Andric, Nebojsa; Ascoli, Mario

    2006-01-01

    Human CG and hFSH elicit a transient increase in ERK1/2 phosphorylation lasting less than 60 min in immature granulosa cells expressing a low density of gonadotropin receptors. In cells expressing a high density of receptors hCG and hFSH elicit this fast transient increase in ERK1/2 phosphorylation and also a delayed and more sustained increase that is detectable after 6–9 h. Both, the early and delayed increases in ERK1/2 phosphorylation can be blocked with inhibitors of PKA, the epidermal growth factor receptor (EGFR) kinase, metalloproteases and MEK. The delayed effect, but not the early effect, can also be blocked with an inhibitor of protein kinase C (PKC). Since the delayed increase in ERK1/2 phosphorylation correlates with low aromatase expression in response to gonadotropins we tested the effects of the inhibitors mentioned on aromatase expression. These inhibitors had little or no effect on gonadotropin-induced aromatase expression in cells expressing a low density of receptors but they enhanced gonadotropin-induced aromatase expression in cells expressing a high density of receptors. Phorbol esters also induced a prolonged increase in ERK1/2 phosphorylation and when added together with hFSH, blocked the induction of aromatase expression by hFSH in cells expressing a low density of hFSHR. A MEK inhibitor reversed the inhibitory effect of the phorbol ester on aromatase induction. We conclude that the effects of gonadotropins on ERK1/2 phosphorylation are mediated by EGF-like growth factors and that the delayed effect is partially mediated by PKC and acts as a negative regulator of aromatase expression. PMID:16973759

  1. Mitochondrial oxidative stress is the achille's heel of melanoma cells resistant to Braf-mutant inhibitor

    PubMed Central

    André, Fanny; Jonneaux, Aurélie; Scalbert, Camille; Garçon, Guillaume; Malet-Martino, Myriam; Balayssac, Stéphane; Rocchi, Stephane; Savina, Ariel; Formstecher, Pierre; Mortier, Laurent; Kluza, Jérome; Marchetti, Philippe

    2013-01-01

    Vemurafenib/PLX4032, a selective inhibitor of mutant BRAFV600E, constitutes a paradigm shift in melanoma therapy. Unfortunately, acquired resistance, which unavoidably occurs, represents one major limitation to clinical responses. Recent studies have highlighted that vemurafenib activated oxidative metabolism in BRAFV600E melanomas expressing PGC1α. However, the oxidative state of melanoma resistant to BRAF inhibitors is unknown. We established representative in vitro and in vivo models of human melanoma resistant to vemurafenib including primary specimens derived from melanoma patients. Firstly, our study reveals that vemurafenib increased mitochondrial respiration and ROS production in BRAFV600E melanoma cell lines regardless the expression of PGC1α. Secondly, melanoma cells that have acquired resistance to vemurafenib displayed intrinsically high rates of mitochondrial respiration associated with elevated mitochondrial oxidative stress irrespective of the presence of vemurafenib. Thirdly, the elevated ROS level rendered vemurafenib-resistant melanoma cells prone to cell death induced by pro-oxidants including the clinical trial drug, elesclomol. Based on these observations, we propose that the mitochondrial oxidative signature of resistant melanoma constitutes a novel opportunity to overcome resistance to BRAF inhibition. PMID:24161908

  2. Evolution of resistance to Aurora kinase B inhibitors in leukaemia cells.

    PubMed

    Failes, Timothy W; Mitic, Gorjana; Abdel-Halim, Heba; Po'uha, Sela T; Liu, Marjorie; Hibbs, David E; Kavallaris, Maria

    2012-01-01

    Aurora kinase inhibitors are new mitosis-targeting drugs currently in clinical trials for the treatment of haematological and solid malignancies. However, knowledge of the molecular factors that influence sensitivity and resistance remains limited. Herein, we developed and characterised an in vitro leukaemia model of resistance to the Aurora B inhibitor ZM447439. Human T-cell acute lymphoblastic leukaemia cells, CCRF-CEM, were selected for resistance in 4 µM ZM447439. CEM/AKB4 cells showed no cross-resistance to tubulin-targeted and DNA-damaging agents, but were hypersensitive to an Aurora kinase A inhibitor. Sequencing revealed a mutation in the Aurora B kinase domain corresponding to a G160E amino acid substitution. Molecular modelling of drug binding in Aurora B containing this mutation suggested that resistance is mediated by the glutamate substitution preventing formation of an active drug-binding motif. Progression of resistance in the more highly selected CEM/AKB8 and CEM/AKB16 cells, derived sequentially from CEM/AKB4 in 8 and 16 µM ZM447439 respectively, was mediated by additional defects. These defects were independent of Aurora B and multi-drug resistance pathways and are associated with reduced apoptosis mostly likely due to reduced inhibition of the catalytic activity of aurora kinase B in the presence of drug. Our findings are important in the context of the use of these new targeted agents in treatment regimes against leukaemia and suggest resistance to therapy may arise through multiple independent mechanisms.

  3. Global Epigenetic Changes Induced by SWI2/SNF2 Inhibitors Characterize Neomycin-Resistant Mammalian Cells

    PubMed Central

    Goswami, Shyamal K.; Komath, Sneha Sudha; Mayo, Marty W.; Hockensmith, Joel W.; Muthuswami, Rohini

    2012-01-01

    Background Previously, we showed that aminoglycoside phosphotransferases catalyze the formation of a specific inhibitor of the SWI2/SNF2 proteins. Aminoglycoside phosphotransferases, for example neomycin-resistant genes, are used extensively as selection markers in mammalian transfections as well as in transgenic studies. However, introduction of the neomycin-resistant gene is fraught with variability in gene expression. We hypothesized that the introduction of neomycin-resistant genes into mammalian cells results in inactivation of SWI2/SNF2 proteins thereby leading to global epigenetic changes. Methodology Using fluorescence spectroscopy we have shown that the inhibitor, known as Active DNA-dependent ATPase A Domain inhibitor (ADAADi), binds to the SWI2/SNF2 proteins in the absence as well as presence of ATP and DNA. This binding occurs via a specific region known as Motif Ia leading to a conformational change in the SWI2/SNF2 proteins that precludes ATP hydrolysis. ADAADi is produced from a plethora of aminoglycosides including G418 and Streptomycin, two commonly used antibiotics in mammalian cell cultures. Mammalian cells are sensitive to ADAADi; however, cells stably transfected with neomycin-resistant genes are refractory to ADAADi. In resistant cells, endogenous SWI2/SNF2 proteins are inactivated which results in altered histone modifications. Microarray data shows that the changes in the epigenome are reflected in altered gene expression. The microarray data was validated using real-time PCR. Finally, we show that the epigenetic changes are quantized. Significance The use of neomycin-resistant genes revolutionized mammalian transfections even though questions linger about efficacy. In this study, we have demonstrated that selection of neomycin-resistant cells results in survival of only those cells that have undergone epigenetic changes, and therefore, data obtained using these resistant genes as selection markers need to be cautiously evaluated. PMID

  4. X-ray structure of human aromatase reveals an androgen-specific active site.

    PubMed

    Ghosh, Debashis; Griswold, Jennifer; Erman, Mary; Pangborn, Walter

    2010-02-28

    Aromatase is a unique cytochrome P450 that catalyzes the removal of the 19-methyl group and aromatization of the A-ring of androgens for the synthesis of estrogens. All human estrogens are synthesized via this enzymatic aromatization pathway. Aromatase inhibitors thus constitute a frontline therapy for estrogen-dependent breast cancer. Despite decades of intense investigation, this enzyme of the endoplasmic reticulum membrane has eluded all structure determination efforts. We have determined the crystal structure of the highly active aromatase purified from human placenta, in complex with its natural substrate androstenedione. The structure shows the binding mode of androstenedione in the catalytically active oxidized high-spin ferric state of the enzyme. Hydrogen bond-forming interactions and tight packing hydrophobic side chains that complement the puckering of the steroid backbone provide the molecular basis for the exclusive androgenic specificity of aromatase. Locations of catalytic residues and water molecules shed new light on the mechanism of the aromatization step. The structure also suggests a membrane integration model indicative of the passage of steroids through the lipid bilayer.

  5. Hypogonadism Associated with Cyp19a1 (Aromatase) Posttranscriptional Upregulation in Celf1 Knockout Mice.

    PubMed

    Boulanger, Gaella; Cibois, Marie; Viet, Justine; Fostier, Alexis; Deschamps, Stéphane; Pastezeur, Sylvain; Massart, Catherine; Gschloessl, Bernhard; Gautier-Courteille, Carole; Paillard, Luc

    2015-09-01

    CELF1 is a multifunctional RNA-binding protein that controls several aspects of RNA fate. The targeted disruption of the Celf1 gene in mice causes male infertility due to impaired spermiogenesis, the postmeiotic differentiation of male gametes. Here, we investigated the molecular reasons that underlie this testicular phenotype. By measuring sex hormone levels, we detected low concentrations of testosterone in Celf1-null mice. We investigated the effect of Celf1 disruption on the expression levels of steroidogenic enzyme genes, and we observed that Cyp19a1 was upregulated. Cyp19a1 encodes aromatase, which transforms testosterone into estradiol. Administration of testosterone or the aromatase inhibitor letrozole partly rescued the spermiogenesis defects, indicating that a lack of testosterone associated with excessive aromatase contributes to the testicular phenotype. In vivo and in vitro interaction assays demonstrated that CELF1 binds to Cyp19a1 mRNA, and reporter assays supported the conclusion that CELF1 directly represses Cyp19a1 translation. We conclude that CELF1 downregulates Cyp19a1 (Aromatase) posttranscriptionally to achieve high concentrations of testosterone compatible with spermiogenesis completion. We discuss the implications of these findings with respect to reproductive defects in men, including patients suffering from isolated hypogonadotropic hypogonadism and myotonic dystrophy type I.

  6. Myeloid cells that impair immunotherapy are restored in melanomas which acquire resistance to BRAF inhibitors.

    PubMed

    Steinberg, Shannon M; Shabaneh, Tamer; Zhang, Peisheng; Martyanov, Viktor; Li, Zhenghui; Malik, Brian; Wood, Tammara; Boni, Andrea; Molodtsov, Aleksey; Angeles, Christina V; Curiel, Tyler J; Whitfield, Michael; Turk, Mary Jo

    2017-02-15

    Acquired resistance to BRAFV600E inhibitors (BRAFi) in melanoma remains a common clinical obstacle, as is the case for any targeted drug therapy that can be developed given the plastic nature of cancers. While there has been significant focus on the cancer cell-intrinsic properties of BRAFi resistance, the impact of BRAFi resistance on host immunity has not been explored. Here we provide preclinical evidence that resistance to BRAFi in an autochthonous mouse model of melanoma is associated with restoration of myeloid-derived suppressor cells (MDSC) in the tumor microenvironment initially reduced by BRAFi treatment. In contrast to restoration of MDSC, levels of T regulatory cells remained reduced in BRAFi-resistant tumors. Accordingly, tumor gene expression signatures specific for myeloid cell chemotaxis and homeostasis reappeared in BRAFi-resistant tumors. Notably, MDSC restoration relied upon MAPK pathway reactivation and downstream production of the myeloid attractant CCL2 in BRAFi-resistant melanoma cells. Strikingly, while combination checkpoint blockade (anti-CTLA-4 + anti-PD-1) was ineffective against BRAFi-resistant melanomas, the addition of MDSC depletion/blockade (anti-Gr-1 + CCR2 antagonist) prevented outgrowth of BRAFi-resistant tumors. Our results illustrate how extrinsic pathways of immunosuppression elaborated by melanoma cells dominate the tumor microenvironment and highlight the need to target extrinsic as well as intrinsic mechanisms of drug resistance.

  7. Drosotoxin, a selective inhibitor of tetrodotoxin-resistant sodium channels.

    PubMed

    Zhu, Shunyi; Gao, Bin; Deng, Meichun; Yuan, Yuzhe; Luo, Lan; Peigneur, Steve; Xiao, Yucheng; Liang, Songping; Tytgat, Jan

    2010-10-15

    The design of animal toxins with high target selectivity has long been a goal in protein engineering. Based on evolutionary relationship between the Drosophila antifungal defensin (drosomycin) and scorpion depressant Na(+) channel toxins, we exploited a strategy to create a novel chimeric molecule (named drosotoxin) with high selectivity for channel subtypes, which was achieved by using drosomycin to substitute the structural core of BmKITc, a depressant toxin acting on both insect and mammalian Na(+) channels. Recombinant drosotoxin selectively inhibited tetrodotoxin-resistant (TTX-R) Na(+) channels in rat dorsal root ganglion (DRG) neurons with a 50% inhibitory concentration (IC(50)) of 2.6+/-0.5muM. This chimeric peptide showed no activity on K(+), Ca(2+) and TTX-sensitive (TTX-S) Na(+) channels in rat DRG neurons and Drosophila para/tipE channels at micromolar concentrations. Drosotoxin represents the first chimeric toxin and example of a non-toxic core scaffold with high selectivity on mammalian TTX-R Na(+) channels.

  8. RAS signaling promotes resistance to JAK inhibitors by suppressing BAD-mediated apoptosis.

    PubMed

    Winter, Peter S; Sarosiek, Kristopher A; Lin, Kevin H; Meggendorfer, Manja; Schnittger, Susanne; Letai, Anthony; Wood, Kris C

    2014-12-23

    Myeloproliferative neoplasms (MPNs) frequently have an activating mutation in the gene encoding Janus kinase 2 (JAK2). Thus, targeting the pathway mediated by JAK and its downstream substrate, signal transducer and activator of transcription (STAT), may yield clinical benefit for patients with MPNs containing the JAK2(V617F) mutation. Although JAK inhibitor therapy reduces splenomegaly and improves systemic symptoms in patients, this treatment does not appreciably reduce the number of neoplastic cells. To identify potential mechanisms underlying this inherent resistance phenomenon, we performed pathway-centric, gain-of-function screens in JAK2(V617F) hematopoietic cells and found that the activation of the guanosine triphosphatase (GTPase) RAS or its effector pathways [mediated by the kinases AKT and ERK (extracellular signal-regulated kinase)] renders cells insensitive to JAK inhibition. Resistant MPN cells became sensitized to JAK inhibitors when also exposed to inhibitors of the AKT or ERK pathways. Mechanistically, in JAK2(V617F) cells, a JAK2-mediated inactivating phosphorylation of the proapoptotic protein BAD [B cell lymphoma 2 (BCL-2)-associated death promoter] promoted cell survival. In sensitive cells, exposure to a JAK inhibitor resulted in dephosphorylation of BAD, enabling BAD to bind and sequester the prosurvival protein BCL-XL (BCL-2-like 1), thereby triggering apoptosis. In resistant cells, RAS effector pathways maintained BAD phosphorylation in the presence of JAK inhibitors, yielding a specific dependence on BCL-XL for survival. In patients with MPNs, activating mutations in RAS co-occur with the JAK2(V617F) mutation in the malignant cells, suggesting that RAS effector pathways likely play an important role in clinically observed resistance.

  9. Uptake inhibitors but not substrates induce protease resistance in extracellular loop two of the dopamine transporter.

    PubMed

    Gaffaney, Jon D; Vaughan, Roxanne A

    2004-03-01

    Changes in protease sensitivity of extracellular loop two (EL2) of the dopamine transporter (DAT) during inhibitor and substrate binding were examined using trypsin proteolysis and epitope-specific immunoblotting. In control rat striatal membranes, proteolysis of DAT in a restricted region of EL2 was produced by 0.001 to 10 microg/ml trypsin. However, in the presence of the dopamine uptake blockers [2-(diphenylmethoxyl) ethyl]-4-(3phenylpropyl) piperazine (GBR 12909), mazindol, 2beta-carbomethoxy-3beta-(4-flourophenyl)tropane (beta-CFT), nomifensine, benztropine, or (-)-cocaine, 100- to 1000-fold higher concentrations of trypsin were required to produce comparable levels of proteolysis. Protease resistance induced by ligands was correlated with their affinity for DAT binding, was not observed with Zn2+, (+)-cocaine, or inhibitors of norepinephrine or serotonin transporters, and was not caused by altered catalytic activity of trypsin. Together, these results support the hypothesis that the interaction of uptake inhibitors with DAT induces a protease-resistant conformation in EL2. In contrast, binding of substrates did not induce protease resistance in EL2, suggesting that substrates and inhibitors interact with DAT differently during binding. To assess the effects of EL2 proteolysis on DAT function, the binding and transport properties of trypsin-digested DAT were assayed with [3H]CFT and [3H]dopamine. Digestion decreased the Bmax for binding and the Vmax for uptake in amounts that were proportional to the extent of proteolysis, indicating that the structural integrity of EL2 is required for maintenance of both DAT binding and transport functions. Together this data provides novel information about inhibitor and substrate interactions at EL2, possibly relating the protease resistant DAT conformation to a mechanism of transport inhibition.

  10. Molecular basis of the inhibition of human aromatase (estrogen synthetase) by flavone and isoflavone phytoestrogens: A site-directed mutagenesis study.

    PubMed

    Kao, Y C; Zhou, C; Sherman, M; Laughton, C A; Chen, S

    1998-02-01

    Flavone and isoflavone phytoestrogens are plant chemicals and are known to be competitive inhibitors of cytochrome P450 aromatase with respect to the androgen substrate. Aromatase is the enzyme that converts androgen to estrogen; therefore, these plant chemicals are thought to be capable of modifying the estrogen level in women. In this study, the inhibition profiles of four flavones [chrysin (5, 7-dihydroxyflavone), 7,8-dihydroxyflavone, baicalein (5,6,7-trihydroxyflavone), and galangin (3,5,7-trihydroxyflavone)], two isoflavones [genistein (4,5,7-trihydroxyisoflavone) and biochanin A (5,7-dihydroxy-4-methoxyisoflavone)], one flavanone [naringenin (4, 5,7-trihydroxyflavanone)], and one naphthoflavone (alpha-naphthoflavone) on the wild-type and six human aromatase mutants (I133Y, P308F, D309A, T310S, I395F, and I474Y) were determined. In combination with computer modeling, the binding characteristics and the structure requirement for flavone and isoflavone phytoestrogens to inhibit human aromatase were obtained. These compounds were found to bind to the active site of aromatase in an orientation in which rings A and C mimic rings D and C of the androgen substrate, respectively. This study also provides a molecular basis as to why isoflavones are significantly poorer inhibitors of aromatase than flavones.

  11. Molecular basis of the inhibition of human aromatase (estrogen synthetase) by flavone and isoflavone phytoestrogens: A site-directed mutagenesis study.

    PubMed Central

    Kao, Y C; Zhou, C; Sherman, M; Laughton, C A; Chen, S

    1998-01-01

    Flavone and isoflavone phytoestrogens are plant chemicals and are known to be competitive inhibitors of cytochrome P450 aromatase with respect to the androgen substrate. Aromatase is the enzyme that converts androgen to estrogen; therefore, these plant chemicals are thought to be capable of modifying the estrogen level in women. In this study, the inhibition profiles of four flavones [chrysin (5, 7-dihydroxyflavone), 7,8-dihydroxyflavone, baicalein (5,6,7-trihydroxyflavone), and galangin (3,5,7-trihydroxyflavone)], two isoflavones [genistein (4,5,7-trihydroxyisoflavone) and biochanin A (5,7-dihydroxy-4-methoxyisoflavone)], one flavanone [naringenin (4, 5,7-trihydroxyflavanone)], and one naphthoflavone (alpha-naphthoflavone) on the wild-type and six human aromatase mutants (I133Y, P308F, D309A, T310S, I395F, and I474Y) were determined. In combination with computer modeling, the binding characteristics and the structure requirement for flavone and isoflavone phytoestrogens to inhibit human aromatase were obtained. These compounds were found to bind to the active site of aromatase in an orientation in which rings A and C mimic rings D and C of the androgen substrate, respectively. This study also provides a molecular basis as to why isoflavones are significantly poorer inhibitors of aromatase than flavones. Images Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 PMID:9435150

  12. Unique Distribution of Aromatase in the Human Brain: In Vivo Studies With PET and [N-Methyl-11C]Vorozole

    SciTech Connect

    Biegon, A.; Biegon, A.; Kim, S.W.; Alexoff, D.; Millard, J.; Carter, P.; Hubbard, B.; King, P.; Logan, J.; Muench, L.; Pareto, D.; Schlyer, D.; Shea, C.; Telang, F.; Wang, G.-J.; Xu, Y.; Fowler, J.

    2010-10-01

    Aromatase catalyzes the last step in estrogen biosynthesis. Brain aromatase is involved in diverse neurophysiological and behavioral functions including sexual behavior, aggression, cognition, and neuroprotection. Using positron emission tomography (PET) with the radiolabeled aromatase inhibitor [N-methyl-{sup 11}C]vorozole, we characterized the tracer distribution and kinetics in the living human brain. Six young, healthy subjects, three men and three women, were administered the radiotracer alone on two separate occasions. Women were scanned in distinct phases of the menstrual cycle. Specificity was confirmed by pretreatment with a pharmacological (2.5 mg) dose of the aromatase inhibitor letrozole. PET data were acquired over a 90-min period and regions of interest placed over selected brain regions. Brain and plasma time activity curves, corrected for metabolites, were used to derive kinetic parameters. Distribution volume (V{sub T}) values in both men and women followed the following rank order: thalamus > amygdala = preoptic area > medulla (inferior olive) > accumbens, pons, occipital and temporal cortex, putamen, cerebellum, and white matter. Pretreatment with letrozole reduced VT in all regions, though the size of the reduction was region-dependent, ranging from {approx}70% blocking in thalamus andpreoptic area to {approx}10% in cerebellum. The high levels of aromatase in thalamus and medulla (inferior olive) appear to be unique to humans. These studies set the stage for the noninvasive assessment of aromatase involvement in various physiological and pathological processes affecting the human brain.

  13. The role of cyclooxygenase-2-dependent signaling via cyclic AMP response element activation on aromatase up-regulation by o,p'-DDT in human breast cancer cells.

    PubMed

    Han, Eun Hee; Kim, Hyung Gyun; Hwang, Yong Pil; Choi, Jae Ho; Im, Ji Hye; Park, Bonghwan; Yang, Ji Hye; Jeong, Tae Cheon; Jeong, Hye Gwang

    2010-10-20

    o,p'-Dichlorodiphenyltrichloroethane (o,p'-DDT) is a DDT isomer and xenoestrogen that can induce inflammation and cancer. However, the effect of o,p'-DDT on aromatase is unclear. Thus, we investigated the effects of o,p'-DDT on aromatase expression in human breast cancer cells. We also examined whether cyclooxygenase-2 (COX-2) is involved in o,p'-DDT-mediated aromatase expression. Treatment with o,p'-DDT-induced aromatase protein expression in MCF-7 and MDA-MB-231 human breast cancer cells; enhancing aromatase gene expression, and enzyme and promoter activity. Treatment with ICI 182.780, a estrogen receptor antagonist, did not affect the inductive effects of o,p'-DDT on aromatase expression. In addition, o,p'-DDT increased COX-2 protein levels markedly, increased COX-2 mRNA expression and promoter activity, enhanced the production of prostaglandin E(2) (PGE(2)), induced cyclic AMP response element (CRE) activation, and cAMP levels and binding of CREB. o,p'-DDT also increased the phosphorylation of PKA, Akt, ERK, and JNK in their signaling pathways in MCF-7 and MDA-MB-231 cells. Finally, o,p'-DDT induction of aromatase was inhibited by various inhibitors [COX-2 (by NS-398), PKA (H-89), PI3-K/Akt (LY 294002), EP2 (AH6809), and EP4 receptor (AH23848)]. Together, these results suggest that o,p'-DDT increases aromatase, and that o,p'-DDT-induced aromatase is correlated with COX-2 up-regulation, mediated via the CRE activation and PKA and PI3-kinase/Akt signaling pathways in breast cancer cells.

  14. Potential effect of Olea europea leaves, Sonchus oleraceus leaves and Mangifera indica peel extracts on aromatase activity in human placental microsomes and CYP19A1 expression in MCF-7 cell line: Comparative study.

    PubMed

    Shaban, N Z; Hegazy, W A; Abdel-Rahman, S M; Awed, O M; Khalil, S A

    2016-08-29

    Aromatase inhibitors (AIs) provide novel approaches to the adjuvant therapy for postmenopausal women with estrogen-receptor-positive (ER+) breast cancers. In this study, different plant extracts from Olea europaea leaves (OLE), Sonchus oleraceus L. (SOE) and Mangifera indica peels (MPE) were prepared to identify phytoconstituents and measure antioxidant capacities. The effects of these three extracts on aromatase activity in human placental microsomes were evaluated. Additionally, the effects of these extracts on tissue-specific promoter expression of CYP19A1 gene in cell culture model (MCF-7) were assessed using qRT-PCR. Results showed a concentration-dependent decrease in aromatase activity after treatment with OLE and MPE, whereas, SOE showed a biphasic effect. The differential effects of OLE, SOE and MPE on aromatase expression showed that OLE seems to be the most potent suppressor followed by SOE and then MPE. These findings indicate that OLE has effective inhibitory action on aromatase at both the enzymatic and expression levels, in addition to its cytotoxic effect against MCF-7 cells. Also, MPE may be has the potential to be used as a tissue-specific aromatase inhibitor (selective aromatase inhibitor) and it may be promising to develop a new therapeutic agent against ER+ breast cancer.

  15. Effects of organochlorine compounds on cytochrome P450 aromatase activity in an immortal sea turtle cell line.

    PubMed

    Keller, Jennifer M; McClellan-Green, Patricia

    2004-01-01

    Many classes of environmental contaminants affect the reproductive function of animals through interactions with the endocrine system. The primary components affected by endocrine active compounds (EACs) are the steroid receptors and the enzymes responsible for steroidogenesis. This study sought to develop an in vitro model for assessing EAC effects in sea turtles by examining their ability to alter cytochrome P450 aromatase (CYP19) activity. Aromatase is the enzyme responsible for the conversion of testosterone to estradiol. This enzyme is critical in the sexual differentiation of reptiles which demonstrate temperature-dependent sex determination. An immortal testis cell line GST-TS from a green sea turtle was grown in culture at 30 degrees C in RPMI 1640 media. The cells were exposed to three known aromatase inducers; dexamethasone (Dex), 8Br-cyclic AMP, or human chronic gonadotropin (HCG) and one aromatase inhibitor 4-androstenol-dione (4-OHA). In addition, the GST-TS cells were exposed to 0.1-30 microM atrazine and 3-100 microM 4,4'-DDE. The inducing compounds that have been shown to increase aromatase activity in other systems failed to induce aromatase activity in the GST-TS cells, yet exposure to the inhibiting compound, 4-OHA, did result in a significant reduction. Atrazine (0.1, 1.0 and 10 microM) significantly induced aromatase activity following a 24 h exposure, and 4,4'-DDE inhibited the activity but only at cytotoxic concentrations (100 microM). Based on these results, this in vitro model can be useful in examining the endocrine effects of EACs in sea turtles.

  16. Resistance Mechanisms for the Bruton’s Tyrosine Kinase Inhibitor Ibrutinib

    PubMed Central

    Woyach, Jennifer A.; Furman, Richard R.; Liu, Ta-Ming; Ozer, Hatice Gulcin; Zapatka, Marc; Ruppert, Amy S.; Xue, Ling; Li, Daniel Hsieh-Hsin; Steggerda, Susanne M.; Versele, Matthias; Dave, Sandeep S.; Zhang, Jenny; Yilmaz, Ayse Selen; Jaglowski, Samantha M.; Blum, Kristie A.; Lozanski, Arletta; Lozanski, Gerard; James, Danelle F.; Barrientos, Jacqueline C.; Lichter, Peter; Stilgenbauer, Stephan; Buggy, Joseph J.; Chang, Betty Y.; Johnson, Amy J.; Byrd, John C.

    2014-01-01

    BACKGROUND Ibrutinib is an irreversible inhibitor of Bruton’s tyrosine kinase (BTK) and is effective in chronic lymphocytic leukemia (CLL). Resistance to irreversible kinase inhibitors and resistance associated with BTK inhibition have not been characterized. Although only a small proportion of patients have had a relapse during ibrutinib therapy, an understanding of resistance mechanisms is important. We evaluated patients with relapsed disease to identify mutations that may mediate ibrutinib resistance. METHODS We performed whole-exome sequencing at baseline and the time of relapse on samples from six patients with acquired resistance to ibrutinib therapy. We then performed functional analysis of identified mutations. In addition, we performed Ion Torrent sequencing for identified resistance mutations on samples from nine patients with prolonged lymphocytosis. RESULTS We identified a cysteine-to-serine mutation in BTK at the binding site of ibrutinib in five patients and identified three distinct mutations in PLCγ2 in two patients. Functional analysis showed that the C481S mutation of BTK results in a protein that is only reversibly inhibited by ibrutinib. The R665W and L845F mutations in PLCγ2 are both potentially gain-of-function mutations that lead to autonomous B-cell–receptor activity. These mutations were not found in any of the patients with prolonged lymphocytosis who were taking ibrutinib. CONCLUSIONS Resistance to the irreversible BTK inhibitor ibrutinib often involves mutation of a cysteine residue where ibrutinib binding occurs. This finding, combined with two additional mutations in PLCγ2 that are immediately downstream of BTK, underscores the importance of the B-cell–receptor pathway in the mechanism of action of ibrutinib in CLL. (Funded by the National Cancer Institute and others.) PMID:24869598

  17. Stat3 contributes to resistance toward BCR-ABL inhibitors in a bone marrow microenvironment model of drug resistance

    PubMed Central

    Bewry, Nadine N.; Nair, Rajesh R.; Emmons, Michael F.; Boulware, David; Pinilla-Ibarz, Javier; Hazlehurst, Lori A.

    2009-01-01

    Imatinib mesylate is a potent, molecularly targeted therapy against the oncogenic tyrosine kinase BCR-ABL. Although imatinib mesylate has considerable efficacy against chronic myeloid leukemia (CML), advanced-stage CML patients frequently become refractory to this agent. The bone marrow is the predominant microenvironment of CML and is a rich source of both soluble factors and extracellular matrices, which may influence drug response. To address the influence of the bone marrow microenvironment on imatinib mesylate sensitivity, we used an in vitro coculture bone marrow stroma model. Our data show culturing K562 cells, in bone marrow stroma-derived conditioned medium (CM), is sufficient to cause resistance to BCR-ABL inhibitors. Drug resistance correlated with increased pTyrStat3, whereas no increases in pTyrStat5 were noted. Moreover, resistance was associated with increased levels of the Stat3 target genes Bcl-xl, Mcl-1, and survivin. Finally, reducing Stat3 levels with small interfering RNA sensitized K562 cells cultured in CM to imatinib mesylate-induced cell death. Importantly, Stat3 dependency was specific for cells grown in CM, as reducing Stat3 levels in regular growth conditions had no effect on imatinib mesylate sensitivity. Together, these data support a novel mechanism of BCR-ABL-independent imatinib mesylate resistance and provide preclinical rationale for using Stat3 inhibitors to increase the efficacy of imatinib mesylate within the context of the bone marrow microenvironment. PMID:18852120

  18. Improving the Resistance Profile of Hepatitis C NS3/4A Inhibitors: Dynamic Substrate Envelope Guided Design

    PubMed Central

    Özen, Ayşegül; Sherman, Woody; Schiffer, Celia A.

    2014-01-01

    Drug resistance is a principal concern in the treatment of quickly evolving diseases. The viral protease NS3/4A is a primary drug target for the hepatitis C virus (HCV) and is known to evolve resistance mutations in response to drug therapy. At the molecular level, drug resistance reflects a subtle change in the balance of molecular recognition by NS3/4A; the drug resistant protease variants are no longer effectively inhibited by the competitive active site inhibitors but can still process the natural substrates with enough efficiency for viral survival. In previous works we have developed the “substrate envelope” hypothesis, which posits that inhibitors should be less susceptible to drug resistance if they better mimic the natural substrate molecular recognition features. In this work, we perform molecular dynamics simulations on four native substrates bound to NS3/4A and discover a clearly conserved dynamic substrate envelope. We show that the most severe drug resistance mutations in NS3/4A occur at residues that are outside the substrate envelope. Comparative analysis of three NS3/4A inhibitors reveals structural and dynamic characteristics of inhibitors that could lead to resistance. We also suggest inhibitor modifications to improve resistance profiles based on the dynamic substrate envelope. This study provides a general framework for guiding the development of novel inhibitors that will be more robust against resistance by mimicking the static and dynamic binding characteristics of natural substrates. PMID:24587770

  19. Histone deacetylase inhibitors: Future therapeutics for insulin resistance and type 2 diabetes.

    PubMed

    Sharma, Sorabh; Taliyan, Rajeev

    2016-11-01

    Insulin resistance is a common feature of obesity and predisposes the affected individuals to a variety of pathologies, including type 2 diabetes mellitus (T2DM), dyslipidemias, hypertension, cardiovascular disease etc. Insulin resistance is the primary cause of T2DM and it occurs many years before the disease onset. Although Thiazolidinediones (TZDs) such as rosiglitazone and pioglitazone are outstanding insulin sensitizers and are in clinical use since 1990s, however, their serious side effects such as heart attack and bladder cancer have limited their utilization. Thus, there is an unmet need to identify a new class of drugs with insulin sensitizing activity and minimal side effects. In the recent years, Histone deacetylase (HDAC) has emerged as a new molecular target in the control of insulin resistance and T2DM. The level of histone acetylation/deacetylation has been found to be altered during insulin resistance and T2DM conditions. HDAC inhibitors have been found to effectively manage insulin resistance and T2DM in various preclinical models and clinical trials. In this review we will focus on various aspects related to regulation of insulin signalling by HDACs and the future scope of HDAC inhibitors as therapeutics for insulin resistance.

  20. Aromatase excess syndrome: a rare autosomal dominant disorder leading to pre- or peri-pubertal onset gynecomastia.

    PubMed

    Fukami, Maki; Miyado, Mami; Nagasaki, Keisuke; Shozu, Makio; Ogata, Tsutomu

    2014-03-01

    Overexpression of CYP19A1 encoding aromatase results in a rare genetic disorder referred to as aromatase excess syndrome (AEXS). Male patients with AEXS manifest pre- or peri-pubertal onset gynecomastia, gonadotropin deficiency, and advanced bone age, while female patients are mostly asymptomatic. To date, 30 male patients with molecularly confirmed AEXS have been reported. A total of 12 types of submicroscopic rearrangements, i.e., two simple duplications, four simple deletions, two simple inversions, and four complex rearrangements, have been implicated in AEXS. Clinical severity of AEXS primarily depends on the types of the rearrangements. AEXS appears to account for a small number of cases of pre- or peri-pubertal onset gynecomastia, and should be suspected particularly when gynecomastia is associated with an autosomal dominant inheritance pattern, characteristic hormone abnormalities and/or advanced bone age. Treatment with an aromatase inhibitor appears to benefit patients with AEXS, although long-term safety of this class of drugs remains unknown.

  1. Sulfonamide-Based Inhibitors of Aminoglycoside Acetyltransferase Eis Abolish Resistance to Kanamycin in Mycobacterium tuberculosis

    SciTech Connect

    Garzan, Atefeh; Willby, Melisa J.; Green, Keith D.; Gajadeera, Chathurada S.; Hou, Caixia; Tsodikov, Oleg V.; Posey, James E.; Garneau-Tsodikova, Sylvie

    2016-12-08

    A two-drug combination therapy where one drug targets an offending cell and the other targets a resistance mechanism to the first drug is a time-tested, yet underexploited approach to combat or prevent drug resistance. By high-throughput screening, we identified a sulfonamide scaffold that served as a pharmacophore to generate inhibitors of Mycobacterium tuberculosis acetyltransferase Eis, whose upregulation causes resistance to the aminoglycoside (AG) antibiotic kanamycin A (KAN) in Mycobacterium tuberculosis. Rational systematic derivatization of this scaffold to maximize Eis inhibition and abolish the Eis-mediated KAN resistance of M. tuberculosis yielded several highly potent agents. A crystal structure of Eis in complex with one of the most potent inhibitors revealed that the inhibitor bound Eis in the AG-binding pocket held by a conformationally malleable region of Eis (residues 28–37) bearing key hydrophobic residues. These Eis inhibitors are promising leads for preclinical development of innovative AG combination therapies against resistant TB.

  2. Search method for inhibitors of Staphyloxanthin production by methicillin-resistant Staphylococcus aureus.

    PubMed

    Sakai, Kent; Koyama, Nobuhiro; Fukuda, Takashi; Mori, Yukiko; Onaka, Hiroyasu; Tomoda, Hiroshi

    2012-01-01

    Staphyloxanthin, a yellow pigment produced by methicillin-resistant Staphylococcus aureus (MRSA), is a virulent factor escaping from the host immune system. A new screening method for inhibitors of staphyloxanthin production by MRSA was established using paper disks. By this screening method, inhibitors of staphyloxanthin production were selected from the natural product library (ca. 300) and from actinomycete culture broths (ca. 1000). From the natural product library, four known inhibitors of lipid metabolism, cerulenin, dihydrobisvertinol, xanthohumol and zaragozic acid, were found to inhibit staphyloxanthin production; however, typical antibiotics used clinically, including vancomycin, had no effect on staphyloxanthin production. From actinomycete culture broths, two known anthraquinones, 6-deoxy-8-O-methylrabelomycin and tetrangomycin, were found to inhibit staphyloxanthin production by MRSA in the paper disk assay. These results suggested that this screening method is useful and effective to find compounds targeting staphyloxanthin production, leading to a new type of chemotherapeutics against MRSA infection.

  3. Potential of Ergosterol Synthesis Inhibitors To Cause Resistance or Cross-Resistance in Trichophyton rubrum

    PubMed Central

    Celandroni, Francesco; Gueye, Sokhna Aissatou; Salvetti, Sara; Senesi, Sonia; Bulgheroni, Anna; Mailland, Federico

    2014-01-01

    Superficial mycoses caused by Trichophyton rubrum are among the most common infections worldwide. T. rubrum infections are difficult to treat and are often associated with recurrences after interruption of the antifungal therapy. Nevertheless, reports on T. rubrum resistance to commonly used antifungal drugs are rare. In this study, we compared the in vitro resistance frequencies and development of resistance to terbinafine, itraconazole, amorolfine, and ciclopirox in T. rubrum. Results demonstrated that naturally occurring mutants were isolated at a frequency of 10−7 for itraconazole and 10−9 for terbinafine and amorolfine. To mimic conditions of body sites in which low drug levels are reached during therapy, T. rubrum was propagated for 10 transfers in media containing subinhibitory drug concentrations. Resistance to itraconazole, terbinafine, and amorolfine emerged at a higher frequency than was seen with spontaneous mutation. Itraconazole-resistant mutants also showed decreased susceptibility to amorolfine as well as to terbinafine, and amorolfine-resistant mutants were also less susceptible to terbinafine. No mutant resistant to ciclopirox was isolated, suggesting no propensity of T. rubrum to develop resistance to this drug. How different drug mechanisms of action can influence the onset of resistance is discussed. PMID:24614379

  4. Aromatase imaging with [N-methyl-C-11]vorozole PET in healthy men and women

    SciTech Connect

    Biegon, Anat; Fowler, Joanna S.; Alexoff, David L.; Kim, Sung Won; Logan, Jean; Pareto, Deborah; Schlyer, David; Wang, Gene-Jack

    2015-02-19

    Aromatase, the last and obligatory enzyme catalyzing estrogen biosynthesis from androgenic precursors, can be labeled in vivo with ¹¹C-vorozole. Aromatase inhibitors are widely used in breast cancer and other endocrine conditions. The present study aims to provide baseline information defining aromatase distribution in healthy men and women, against which its perturbation in pathological situations can be studied. Methods: ¹¹C-vorozole (111-296 MBq/subject) was injected I.V in 13 men and 20 women (age range 23 to 67). PET data were acquired over a 90 minute period. Each subject had 4 scans, 2/day separated by 2-6 weeks, including brain and torso or pelvis scans. Young women were scanned at 2 discrete phases of the menstrual cycle (midcycle and late luteal). Men and postmenopausal women were also scanned following pretreatment with a clinical dose of the aromatase inhibitor letrozole (“blocking” studies). Time activity curves were obtained and standard uptake values (SUV) calculated for major organs including brain, heart, lungs, liver, kidneys, spleen, muscle, bone and male and female reproductive organs (penis, testes, uterus, ovaries). Organ and whole body radiation exposures were calculated using Olinda software. Results: Liver uptake was higher than all other organs, but was not blocked by pretreatment with letrozole. Mean SUVs in men were higher than in women, and brain uptake was blocked by letrozole. Male brain SUVs were also higher than all other organs (ranging from 0.48±0.05 in lungs to 1.5±0.13 in kidneys). Mean ovarian SUVs (3.08±0.7) were comparable to brain levels and higher than all other organs. Furthermore, ovarian SUVs In young women around the time of ovulation (midcycle) were significantly higher than those measured in the late luteal phase, while aging and cigarette smoking reduced ¹¹C-vorozole uptake. Conclusions: PET with ¹¹C-vorozole is useful for assessing physiological changes in estrogen synthesis capacity in the human body

  5. Aromatase imaging with [N-methyl-C-11]vorozole PET in healthy men and women

    DOE PAGES

    Biegon, Anat; Fowler, Joanna S.; Alexoff, David L.; ...

    2015-02-19

    Aromatase, the last and obligatory enzyme catalyzing estrogen biosynthesis from androgenic precursors, can be labeled in vivo with ¹¹C-vorozole. Aromatase inhibitors are widely used in breast cancer and other endocrine conditions. The present study aims to provide baseline information defining aromatase distribution in healthy men and women, against which its perturbation in pathological situations can be studied. Methods: ¹¹C-vorozole (111-296 MBq/subject) was injected I.V in 13 men and 20 women (age range 23 to 67). PET data were acquired over a 90 minute period. Each subject had 4 scans, 2/day separated by 2-6 weeks, including brain and torso or pelvismore » scans. Young women were scanned at 2 discrete phases of the menstrual cycle (midcycle and late luteal). Men and postmenopausal women were also scanned following pretreatment with a clinical dose of the aromatase inhibitor letrozole (“blocking” studies). Time activity curves were obtained and standard uptake values (SUV) calculated for major organs including brain, heart, lungs, liver, kidneys, spleen, muscle, bone and male and female reproductive organs (penis, testes, uterus, ovaries). Organ and whole body radiation exposures were calculated using Olinda software. Results: Liver uptake was higher than all other organs, but was not blocked by pretreatment with letrozole. Mean SUVs in men were higher than in women, and brain uptake was blocked by letrozole. Male brain SUVs were also higher than all other organs (ranging from 0.48±0.05 in lungs to 1.5±0.13 in kidneys). Mean ovarian SUVs (3.08±0.7) were comparable to brain levels and higher than all other organs. Furthermore, ovarian SUVs In young women around the time of ovulation (midcycle) were significantly higher than those measured in the late luteal phase, while aging and cigarette smoking reduced ¹¹C-vorozole uptake. Conclusions: PET with ¹¹C-vorozole is useful for assessing physiological changes in estrogen synthesis capacity in the

  6. [Bacterial efflux pumps - their role in antibiotic resistance and potential inhibitors].

    PubMed

    Hricová, Kristýna; Kolář, Milan

    2014-12-01

    Efflux pumps capable of actively draining antibiotic agents from bacterial cells may be considered one of potential mechanisms of the development of antimicrobial resistance. The most important group of efflux pumps capable of removing several types of antibiotics include RND (resistance - nodulation - division) pumps. These are three proteins that cross the bacterial cell wall, allowing direct expulsion of the agent out from the bacterial cell. The most investigated efflux pumps are the AcrAB-TolC system in Escherichia coli and the MexAB-OprM system in Pseudomonas aeruginosa. Moreover, efflux pumps are able to export other than antibacterial agents such as disinfectants, thus decreasing their effectiveness. One potential approach to inactivation of an efflux pump is to use the so-called efflux pump inhibitors (EPIs). Potential inhibitors tested in vitro involve, for example, phenylalanyl-arginyl-b-naphthylamide (PAbN), carbonyl cyanide m-chlorophenylhydrazone (CCCP) or agents of the phenothiazine class.

  7. Resistance of Hepatitis C Virus to Inhibitors: Complexity and Clinical Implications

    PubMed Central

    Perales, Celia; Quer, Josep; Gregori, Josep; Esteban, Juan Ignacio; Domingo, Esteban

    2015-01-01

    Selection of inhibitor-resistant viral mutants is universal for viruses that display quasi-species dynamics, and hepatitis C virus (HCV) is no exception. Here we review recent results on drug resistance in HCV, with emphasis on resistance to the newly-developed, directly-acting antiviral agents, as they are increasingly employed in the clinic. We put the experimental observations in the context of quasi-species dynamics, in particular what the genetic and phenotypic barriers to resistance mean in terms of exploration of sequence space while HCV replicates in the liver of infected patients or in cell culture. Strategies to diminish the probability of viral breakthrough during treatment are briefly outlined. PMID:26561827

  8. EAI045: The fourth-generation EGFR inhibitor overcoming T790M and C797S resistance.

    PubMed

    Wang, Shuhang; Song, Yongping; Liu, Delong

    2017-01-28

    The third-generation tyrosine kinase inhibitors (TKI), AZD9291 (osimertinib) and CO-1686 (rociletinib) of epidermal growth factor receptor (EGFR) are highly active against T790M positive non-small cell lung cancer (NSCLC). However, resistance develops rapidly. EGFR C797S mutation was reported to be a leading mechanism of resistance to the third-generation inhibitors. The C797S mutation appears to be an ideal target for overcoming the acquired resistance to the third-generation inhibitors. This review summarizes the latest development on the discovery of a fourth-generation EGFR TKI, EAI045.3.

  9. Overcoming resistance of glioblastoma to conventional cytotoxic therapies by the addition of PARP inhibitors.

    PubMed

    Chalmers, Anthony J

    2010-09-01

    This article will present the rationale for combining chemical inhibitors of the DNA repair enzyme poly(ADP-ribose) polymerase (PARP) with conventional cytotoxic agents to improve the treatment of glioblastoma. After a brief review of the current therapeutic options for these aggressive tumours, the possible reasons for their resistance to radiation and chemotherapy will be discussed, highlighting the important role of DNA damage response pathways in many key resistance mechanisms. The dose-limiting toxicities associated with radiation and chemotherapy treatment will be described in order to illustrate the importance of tumour specificity in any attempt to increase the effectiveness of conventional treatments. There will then be a summary of the reasons why targeting DNA repair pathways might achieve tumour specific sensitization. After a brief summary of the key DNA damage response pathways, the biology, biochemistry and pharmacology of PARP and the existing PARP inhibitors will be presented. The major part of the review will cover the effects of combining PARP inhibitors with radiation and chemotherapy in vitro and in vivo, commenting on the underlying mechanisms and indicating where the data are predictive of tumour specific sensitization. Finally, we will consider specific scenarios where PARP inhibitors might contribute to the treatment of glioblastoma patients, discuss the challenges and opportunities associated with early phase clinical testing of these agents, and describe the clinical trials that are either underway or in development.

  10. Structures of HIV Protease Guide Inhibitor Design to Overcome Drug Resistance

    SciTech Connect

    Weber, Irene T.; Kovalevsky, Andrey Y.; Harrison, Robert W.

    2008-06-03

    The HIV/AIDS infection continues to be a major epidemic worldwide despite the initial promise of antiviral drugs. Current therapy includes a combination of drugs that inhibit two of the virally-encoded enzymes, the reverse transcriptase and the protease. The first generation of HIV protease inhibitors that have been in clinical use for treatment of AIDS since 1995 was developed with the aid of structural analysis of protease-inhibitor complexes. These drugs were successful in improving the life span of HIV-infected people. Subsequently, the rapid emergence of drug resistance has necessitated the design of new inhibitors that target mutant proteases. This second generation of antiviral protease inhibitors has been developed with the aid of data from medicinal chemistry, kinetics, and X-ray crystallographic analysis. Traditional computational methods such as molecular mechanics and dynamics can be supplemented with intelligent data mining approaches. One approach, based on similarities to the protease interactions with substrates, is to incorporate additional interactions with main chain atoms that cannot easily be eliminated by mutations. Our structural and inhibition data for darunavir have helped to understand its antiviral activity and effectiveness on drug resistant HIV and demonstrate the success of this approach.

  11. New HSP27 inhibitors efficiently suppress drug resistance development in cancer cells

    PubMed Central

    Lennig, Petra; Zhang, Yixin; Schroeder, Michael

    2016-01-01

    Drug resistance is an important open problem in cancer treatment. In recent years, the heat shock protein HSP27 (HSPB1) was identified as a key player driving resistance development. HSP27 is overexpressed in many cancer types and influences cellular processes such as apoptosis, DNA repair, recombination, and formation of metastases. As a result cancer cells are able to suppress apoptosis and develop resistance to cytostatic drugs. To identify HSP27 inhibitors we follow a novel computational drug repositioning approach. We exploit a similarity between a predicted HSP27 binding site to a viral thymidine kinase to generate lead inhibitors for HSP27. Six of these leads were verified experimentally. They bind HSP27 and down-regulate its chaperone activity. Most importantly, all six compounds inhibit development of drug resistance in cellular assays. One of the leads – chlorpromazine – is an antipsychotic, which has a positive effect on survival time in human breast cancer. In summary, we make two important contributions: First, we put forward six novel leads, which inhibit HSP27 and tackle drug resistance. Second, we demonstrate the power of computational drug repositioning. PMID:27626687

  12. Response and resistance to BET bromodomain inhibitors in triple negative breast cancer

    PubMed Central

    Tabassum, Doris P.; Roberts, Justin M.; Janiszewska, Michalina; Huh, Sung Jin; Liang, Yi; Ryan, Jeremy; Doherty, Ernest; Mohammed, Hisham; Guo, Hao; Stover, Daniel G.; Ekram, Muhammad B.; Brown, Jonathan; D'Santos, Clive; Krop, Ian E.; Dillon, Deborah; McKeown, Michael; Ott, Christopher; Qi, Jun; Ni, Min; Rao, Prakash K.; Duarte, Melissa; Wu, Shwu-Yuan; Chiang, Cheng-Ming; Anders, Lars; Young, Richard A.; Winer, Eric; Letai, Antony; Barry, William T.; Carroll, Jason S.; Long, Henry; Brown, Myles; Liu, X. Shirley; Meyer, Clifford A.; Bradner, James E.; Polyak, Kornelia

    2015-01-01

    Triple negative breast cancer (TNBC) is a heterogeneous and clinically aggressive disease for which there is no targeted therapy1-3. BET bromodomain inhibitors, which have shown efficacy in several models of cancer4-6, have not been evaluated in TNBC. These inhibitors displace BET bromodomain proteins such as BRD4 from chromatin by competing with their acetyllysine recognition modules, leading to inhibition of oncogenic transcriptional programs7-9. Here we report the preferential sensitivity of TNBCs to BET bromodomain inhibition in vitro and in vivo, establishing a rationale for clinical investigation and further motivation to understand mechanisms of resistance. In paired cell lines selected for acquired resistance to BET inhibition from previously sensitive TNBCs, we failed to identify gatekeeper mutations, new driver events or drug pump activation. BET-resistant TNBC cells remain dependent on wild-type BRD4, which supports transcription and cell proliferation in a bromodomain-independent manner. Proteomic studies of resistant TNBC identify strong association with MED1 and hyper-phosphorylation of BRD4 attributable to decreased activity of PP2A, identified here as a principal BRD4 serine phosphatase. Together, these studies provide a rationale for BET inhibition in TNBC and present mechanism-based combination strategies to anticipate clinical drug resistance. PMID:26735014

  13. QSAR modeling of aromatase inhibitory activity of 1-substituted 1,2,3-triazole analogs of letrozole.

    PubMed

    Nantasenamat, Chanin; Worachartcheewan, Apilak; Prachayasittikul, Supaluk; Isarankura-Na-Ayudhya, Chartchalerm; Prachayasittikul, Virapong

    2013-11-01

    Aromatase is an estrogen biosynthesis enzyme belonging to the cytochrome P450 family that catalyzes the rate-limiting step of converting androgens to estrogens. As it is pertinent toward tumor cell growth promotion, aromatase is a lucrative therapeutic target for breast cancer. In the pursuit of robust aromatase inhibitors, a set of fifty-four 1-substituted mono- and bis-benzonitrile or phenyl analogs of 1,2,3-triazole letrozole were employed in quantitative structure-activity relationship (QSAR) study using multiple linear regression (MLR), artificial neural network (ANN) and support vector machine (SVM). Such QSAR models were developed using a set of descriptors providing coverage of the general characteristics of a molecule encompassing molecular size, flexibility, polarity, solubility, charge and electronic properties. Important physicochemical properties giving rise to good aromatase inhibition were obtained by means of exploring its chemical space as a function of the calculated molecular descriptors. The optimal subset of 3 descriptors (i.e. number of rings, ALogP and HOMO-LUMO) was further used for QSAR model construction. The predicted pIC₅₀ values were in strong correlation with their experimental values displaying correlation coefficient values in the range of 0.72-0.83 for the cross-validated set (QCV) while the external test set (Q(Ext)) afforded values in the range of 0.65-0.66. Insights gained from the present study are anticipated to provide pertinent information contributing to the origins of aromatase inhibitory activity and therefore aid in our on-going quest for aromatase inhibitors with robust properties.

  14. The effect of opiates on the activity of human placental aromatase/CYP19.

    PubMed

    Zharikova, Olga L; Deshmukh, Sujal V; Kumar, Meena; Vargas, Ricardo; Nanovskaya, Tatiana N; Hankins, Gary D V; Ahmed, Mahmoud S

    2007-01-15

    Aromatase, cytochrome P450 19, is a key enzyme in the biosynthesis of estrogens by the human placenta. It is also the major placental enzyme that metabolizes the opiates L-acetylmethadol (LAAM), methadone, and buprenorphine (BUP). Methadone and BUP are used in treatment of the opiate addict and are competitive inhibitors of testosterone conversion to estradiol (E(2)) and 16alpha-hydroxytestosterone (16-OHT) to estriol (E(3)) by aromatase. The aim of this investigation is to determine the effect of 20 opiates, which can be administered to pregnant patients for therapeutic indications or abused, on E(2) and E(3) formation by placental aromatase. Data obtained indicated that the opiates increased, inhibited, or had no effect on aromatase activity. Their effect on E(3) formation was more pronounced than that on E(2) due to the lower affinity of 16-OHT than testosterone to aromatase. The K(i) values for the opiates that inhibited E(3) formation were sufentanil, 7 +/- 1 microM; LAAM, 13 +/- 8 microM; fentanyl, 25 +/- 5 microM; oxycodone, 92 +/- 22 microM; codeine, 218 +/- 69 microM; (+)-pentazocine, 225 +/- 73 microM. The agonists morphine, heroin, hydromorphone, oxymorphone, hydrocodone, propoxyphene, meperidine, levorphanol, dextrorphan, and (-)-pentazocine and the antagonists naloxone and naltrexone caused an increase in E(3) formation by 124-160% of control but had no effect on E(2) formation. Moreover, oxycodone and codeine did not inhibit E(2) formation and the IC(50) values for fentanyl, sufentanil, and (+)-pentazocine were >1000 microM. It is unlikely that the acute administration of the opiates that inhibit estrogen formation would affect maternal and/or neonatal outcome. However, the effects of abusing any of them during the entire pregnancy are unclear at this time.

  15. Placental expression and molecular characterization of aromatase cytochrome P450 in the spotted hyena (Crocuta crocuta).

    PubMed

    Conley, A J; Corbin, C J; Browne, P; Mapes, S M; Place, N J; Hughes, A L; Glickman, S E

    2007-07-01

    At birth, the external genitalia of female spotted hyenas (Crocuta crocuta) are the most masculinized of any known mammal, but are still sexually differentiated. Placental aromatase cytochrome P450 (P450arom) is an important route of androgen metabolism protecting human female fetuses from virilization in utero. Therefore, placental P450arom expression was examined in spotted hyenas to determine levels during genital differentiation, and to compare molecular characteristics between the hyena and human placental enzymes. Hyena placental P450arom activity was determined at gestational days (GD) 31, 35, 45, 65 and 95 (term, 110), and the relative sensitivity of hyena and human placental enzyme to inhibition by the specific inhibitor, Letrozole, was also examined. Expression of hyena P450arom in placenta was localized by immuno-histochemistry, and a full-length cDNA was cloned for phylogenetic analysis. Aromatase activity increased from GD31 to a peak at 45 and 65, apparently decreasing later in gestation. This activity was more sensitive to inhibition by Letrozole than was human placental aromatase activity. Expression of P450arom was localized to syncytiotrophoblast and giant cells of mid-gestation placentas. The coding sequence of hyena P450arom was 94% and 86% identical to the canine and human enzymes respectively, as reflected by phylogenetic analyses. These data demonstrate for the first time that hyena placental aromatase activity is comparable to that of human placentas when genital differentiation is in progress. This suggests that even in female spotted hyenas clitoral differentiation is likely protected from virilization by placental androgen metabolism. Decreased placental aromatase activity in late gestation may be equally important in allowing androgen to program behaviors at birth. Although hyena P450arom is closely related to the canine enzyme, both placental anatomy and P450arom expression differ. Other hyaenids and carnivores must be investigated to

  16. Improvement of the linear polarization resistance method for testing steel corrosion inhibitors

    NASA Astrophysics Data System (ADS)

    Faritov, A. T.; Rozhdestvenskii, Yu. G.; Yamshchikova, S. A.; Minnikhanova, E. R.; Tyusenkov, A. S.

    2016-11-01

    The linear polarization resistance method is used to improve the technique of corrosion control in liquid conducting according to GOST 9.514-99 (General Corrosion and Aging Protection System. Corrosion Inhibitors for Metals in Water Systems. Electrochemical Method of Determining the Protective Ability). Corrosion monitoring is shown to be performed by electronic devices with real-time data transfer to industrial controllers and SCADA systems.

  17. Targeting abnormal DNA double strand break repair in tyrosine kinase inhibitor-resistant chronic myeloid leukemias

    PubMed Central

    Tobin, Lisa A.; Robert, Carine; Rapoport, Aaron P.; Gojo, Ivana; Baer, Maria R.; Tomkinson, Alan E.; Rassool, Feyruz V.

    2013-01-01

    Resistance to imatinib (IM) and other BCR-ABL1 tyrosine kinase inhibitors (TKI)s is an increasing problem in leukemias caused by expression of BCR-ABL1. Since chronic myeloid leukemia (CML) cell lines expressing BCR-ABL1 utilize an alternative non-homologous end-joining pathway (ALT NHEJ) to repair DNA double strand breaks (DSB)s, we asked whether this repair pathway is a novel therapeutic target in TKI-resistant disease. Notably, the steady state levels of two ALT NHEJ proteins, poly-(ADP-ribose) polymerase 1 (PARP1) and DNA ligase IIIα were increased in the BCR-ABL1-positive CML cell line K562 and, to a greater extent, in its imatinib resistant (IMR) derivative. Incubation of these cell lines with a combination of DNA ligase and PARP inhibitors inhibited ALT NHEJ and selectively decreased survival with the effect being greater in the IMR derivative. Similar results were obtained with TKI-resistant derivatives of two hematopoietic cell lines that had been engineered to stably express BCR-ABL1. Together our results show that the sensitivity of cell lines expressing BCR-ABL1 to the combination of DNA ligase and PARP inhibitors correlates with the steady state levels of PARP1 and DNA ligase IIIα, and ALT NHEJ activity. Importantly, analysis of clinical samples from CML patients confirmed that the expression levels of PARP1 and DNA ligase IIIα correlated with sensitivity to the DNA repair inhibitor combination. Thus, the expression levels of PARP1 and DNA ligase IIIα serve as biomarkers to identify a subgroup of CML patients who may be candidates for therapies that target the ALT NHEJ pathway when treatment with TKIs has failed. PMID:22641215

  18. Triclosan Derivatives: Towards Potent Inhibitors of Drug-Sensitive and Drug-Resistant Mycobacterium tuberculosis

    SciTech Connect

    Freundlich, Joel S.; Wang, Feng; Vilchèze, Catherine; Gulten, Gulcin; Langley, Robert; Schiehser, Guy A.; Jacobus, David P.; Jacobs, Jr., William R.; Sacchettini, James C.

    2009-06-30

    Isoniazid (INH) is a frontline antitubercular drug that inhibits the enoyl acyl carrier protein reductase InhA. Novel inhibitors of InhA that are not cross-resistant to INH represent a significant goal in antitubercular chemotherapy. The design, synthesis, and biological activity of a series of triclosan-based inhibitors is reported, including their promising efficacy against INH-resistant strains of M. tuberculosis. Triclosan has been previously shown to inhibit InhA, an essential enoyl acyl carrier protein reductase involved in mycolic acid biosynthesis, the inhibition of which leads to the lysis of Mycobacterium tuberculosis. Using a structure-based drug design approach, a series of 5-substituted triclosan derivatives was developed. Two groups of derivatives with alkyl and aryl substituents, respectively, were identified with dramatically enhanced potency against purified InhA. The most efficacious inhibitor displayed an IC{sub 50} value of 21 nM, which was 50-fold more potent than triclosan. X-ray crystal structures of InhA in complex with four triclosan derivatives revealed the structural basis for the inhibitory activity. Six selected triclosan derivatives were tested against isoniazid-sensitive and resistant strains of M. tuberculosis. Among those, the best inhibitor had an MIC value of 4.7 {mu}g mL{sup -1} (13 {mu}M), which represents a tenfold improvement over the bacteriocidal activity of triclosan. A subset of these triclosan analogues was more potent than isoniazid against two isoniazid-resistant M. tuberculosis strains, demonstrating the significant potential for structure-based design in the development of next generation antitubercular drugs.

  19. Novel Mechanisms of PARP Inhibitor Resistance in BRCA1-Deficient Breast Cancers

    DTIC Science & Technology

    2015-12-01

    of single stranded DNA breaks especially through activation of base excision repair (BER) (Krishnakumar & Kraus, 2010). A synthetic lethal phenotype...1) Test candidate mechanisms of HR restoration: a) Determine HR activity in parental , rescue, and PARP inhibitor resistant lines: In my previous two...seen in the parental , BRCA1-deficient line using Rad51 and PALB2 as markers. b) Test BRCA1-restoration: In my previous annual reports, I showed that

  20. Colocalization of aromatase in spinal cord astrocytes: Differences in expression and relationship to mechanical and thermal hyperalgesia in murine models of a painful and a non-painful bone tumor

    PubMed Central

    O’Brien, Elaine E; Smeester, Branden A; Michlitsch, Kyle S; Lee, Jang-Hern; Beitz, Alvin J

    2015-01-01

    While spinal cord astrocytes play a key role in the generation of cancer pain, there have been no studies that have examined the relationship of tumor-induced astrocyte activation and aromatase expression during the development of cancer pain. Here, we examined tumor-induced mechanical hyperalgesia and cold allodynia, and changes in GFAP and aromatase expression in murine models of painful and non-painful bone cancer. We demonstrate that implantation of fibrosarcoma cells, but not melanoma cells, produces robust mechanical hyperalgesia and cold allodynia in tumor-bearing mice compared to saline-injected controls. Secondly, this increase in mechanical hyperalgesia and cold allodynia is mirrored by significant increases in both spinal astrocyte activity and aromatase expression in the dorsal horn of fibrosarcoma-bearing mice. Importantly, we show that aromatase is only found within a subset of astrocytes and not in neurons in the lumbar spinal cord. Finally, administration of an aromatase inhibitor reduced tumor-induced hyperalgesia in fibrosarcoma-bearing animals. We conclude that a painful fibrosarcoma tumor induces a significant increase in spinal astrocyte activation and aromatase expression and that the up-regulation of aromatase plays a role in the development of bone tumor-induced hyperalgesia. Since spinal aromatase is also upregulated, but to a lesser extent, in non-painful melanoma bone tumors, it may also be neuroprotective and responsive to the changing tumor environment. PMID:26071956

  1. Neutralizing antibody and anti-retroviral drug sensitivities of HIV-1 isolates resistant to small molecule CCR5 inhibitors

    SciTech Connect

    Pugach, Pavel; Ketas, Thomas J.; Michael, Elizabeth; Moore, John P.

    2008-08-01

    The small molecule CCR5 inhibitors are a new class of drugs for treating infection by human immunodeficiency virus type 1 (HIV-1). They act by binding to the CCR5 co-receptor and preventing its use during HIV-1-cell fusion. Escape mutants can be raised against CCR5 inhibitors in vitro and will arise when these drugs are used clinically. Here, we have assessed the responses of CCR5 inhibitor-resistant viruses to other anti-retroviral drugs that act by different mechanisms, and their sensitivities to neutralizing antibodies (NAbs). The rationale for the latter study is that the resistance pathway for CCR5 inhibitors involves changes in the HIV-1 envelope glycoproteins (Env), which are also targets for NAbs. The escape mutants CC101.19 and D1/85.16 were selected for resistance to AD101 and vicriviroc (VVC), respectively, from the primary R5 HIV-1 isolate CC1/85. Each escape mutant was cross-resistant to other small molecule CCR5 inhibitors (aplaviroc, maraviroc, VVC, AD101 and CMPD 167), but sensitive to protein ligands of CCR5: the modified chemokine PSC-RANTES and the humanized MAb PRO-140. The resistant viruses also retained wild-type sensitivity to the nucleoside reverse transcriptase inhibitor (RTI) zidovudine, the non-nucleoside RTI nevirapine, the protease inhibitor atazanavir and other attachment and fusion inhibitors that act independently of CCR5 (BMS-806, PRO-542 and enfuvirtide). Of note is that the escape mutants were more sensitive than the parental CC1/85 isolate to a subset of neutralizing monoclonal antibodies and to some sera from HIV-1-infected people, implying that sequence changes in Env that confer resistance to CCR5 inhibitors can increase the accessibility of some NAb epitopes. The need to preserve NAb resistance may therefore be a constraint upon how escape from CCR5 inhibitors occurs in vivo.

  2. Neutralizing antibody and anti-retroviral drug sensitivities of HIV-1 isolates resistant to small molecule CCR5 inhibitors

    PubMed Central

    Pugach, Pavel; Ketas, Thomas J.; Michael, Elizabeth; Moore, John P.

    2008-01-01

    The small molecule CCR5 inhibitors are a new class of drugs for treating infection by human immunodeficiency virus type 1 (HIV-1). They act by binding to the CCR5 co-receptor and preventing its use during HIV-1-cell fusion. Escape mutants can be raised against CCR5 inhibitors in vitro and will arise when these drugs are used clinically. Here, we have assessed the responses of CCR5 inhibitor-resistant viruses to other anti-retroviral drugs that act by different mechanisms, and their sensitivities to neutralizing antibodies (NAbs). The rationale for the latter study is that the resistance pathway for CCR5 inhibitors involves changes in the HIV-1 envelope glycoproteins (Env), which are also targets for NAbs. The escape mutants CC101.19 and D1/85.16 were selected for resistance to AD101 and vicriviroc (VVC), respectively, from the primary R5 HIV-1 isolate CC1/85. Each escape mutant was cross resistant to other small molecule CCR5 inhibitors (aplaviroc, maraviroc, VVC, AD101 and CMPD 167), but sensitive to protein ligands of CCR5: the modified chemokine PSC-RANTES and the humanized MAb PRO 140. The resistant viruses also retained wild-type sensitivity to the nucleoside reverse transcriptase inhibitor (RTI) zidovudine, the non-nucleoside RTI nevirapine, the protease inhibitor atazanavir and other attachment and fusion inhibitors that act independently of CCR5 (BMS-806, PRO-542 and enfuvirtide). Of note is that the escape mutants were more sensitive than the parental CC1/85 isolate to a subset of neutralizing monoclonal antibodies and to some sera from HIV-1-infected people, implying that sequence changes in Env that confer resistance to CCR5 inhibitors can increase the accessibility of some NAb epitopes. The need to preserve NAb resistance may therefore be a constraint upon how escape from CCR5 inhibitors occurs in vivo. PMID:18519143

  3. Rapid emergence of hepatitis C virus protease inhibitor resistance is expected

    SciTech Connect

    Rong, Libin; Perelson, Alan S; Ribeiro, Ruy M

    2009-01-01

    Approximately 170 million people worldwide are infected with hepatitis C virus (HCV). Current therapy, consisting of pegylated interferon (PEG-IFN) and ribavirin (RBV), leads to sustained viral elimination in only about 45% of patients treated. Telaprevir (VX-950), a novel HCV NS3-4A serine protease inhibitor, has demonstrated substantial antiviral activity in patients with chronic hepatitis C genotype 1 infection. However, some patients experience viral breakthrough during dosing, with drug resistant variants being 5%-20% of the virus population as early as day 2 after treatment initiation. Why viral variants appear such a short time after the start of dosing is unclear, especially since this has not been seen with monotherapy for either human immunodeficiency virus or hepatitis B virus. Here, using a viral dynamic model, we explain why such rapid emergence of drug resistant variants is expected when potent HCV protease inhibitors are used as monotherapy. Surprisingly, our model also shows that such rapid emergence need not be the case with some potent HCV NS5B polymerase inhibitors. Examining the case of telaprevir therapy in detail, we show the model fits observed dynamics of both wild-type and drug-resistant variants during treatment, and supports combination therapy of direct antiviral drugs with PEG-IFN and/or RBV for hepatitis C.

  4. Biochemical, inhibition and inhibitor resistance studies of xenotropic murine leukemia virus-related virus reverse transcriptase

    PubMed Central

    Ndongwe, Tanyaradzwa P.; Adedeji, Adeyemi O.; Michailidis, Eleftherios; Ong, Yee Tsuey; Hachiya, Atsuko; Marchand, Bruno; Ryan, Emily M.; Rai, Devendra K.; Kirby, Karen A.; Whatley, Angela S.; Burke, Donald H.; Johnson, Marc; Ding, Shilei; Zheng, Yi-Min; Liu, Shan-Lu; Kodama, Ei-Ichi; Delviks-Frankenberry, Krista A.; Pathak, Vinay K.; Mitsuya, Hiroaki; Parniak, Michael A.; Singh, Kamalendra; Sarafianos, Stefan G.

    2012-01-01

    We report key mechanistic differences between the reverse transcriptases (RT) of human immunodeficiency virus type-1 (HIV-1) and of xenotropic murine leukemia virus-related virus (XMRV), a gammaretrovirus that can infect human cells. Steady and pre-steady state kinetics demonstrated that XMRV RT is significantly less efficient in DNA synthesis and in unblocking chain-terminated primers. Surface plasmon resonance experiments showed that the gammaretroviral enzyme has a remarkably higher dissociation rate (koff) from DNA, which also results in lower processivity than HIV-1 RT. Transient kinetics of mismatch incorporation revealed that XMRV RT has higher fidelity than HIV-1 RT. We identified RNA aptamers that potently inhibit XMRV, but not HIV-1 RT. XMRV RT is highly susceptible to some nucleoside RT inhibitors, including Translocation Deficient RT inhibitors, but not to non-nucleoside RT inhibitors. We demonstrated that XMRV RT mutants K103R and Q190M, which are equivalent to HIV-1 mutants that are resistant to tenofovir (K65R) and AZT (Q151M), are also resistant to the respective drugs, suggesting that XMRV can acquire resistance to these compounds through the decreased incorporation mechanism reported in HIV-1. PMID:21908397

  5. Novel β-lactamase inhibitors: a therapeutic hope against the scourge of multidrug resistance

    PubMed Central

    Watkins, Richard R.; Papp-Wallace, Krisztina M.; Drawz, Sarah M.; Bonomo, Robert A.

    2013-01-01

    The increasing incidence and prevalence of multi-drug resistance (MDR) among contemporary Gram-negative bacteria represents a significant threat to human health. Since their discovery, β-lactam antibiotics have been a major component of the armamentarium against these serious pathogens. Unfortunately, a wide range of β-lactamase enzymes have emerged that are capable of inactivating these powerful drugs. In the past 30 years, a major advancement in the battle against microbes has been the development of β-lactamase inhibitors, which restore the efficacy of β-lactam antibiotics (e.g., ampicillin/sulbactam, amoxicillin/clavulanate, ticarcillin/clavulanate, and piperacillin/tazobactam). Unfortunately, many newly discovered β-lactamases are not inactivated by currently available inhibitors. Is there hope? For the first time in many years, we can anticipate the development and introduction into clinical practice of novel inhibitors. Although these inhibitors may still not be effective for all β-lactamases, their introduction is still welcome. This review focuses on the novel β-lactamase inhibitors that are closest to being introduced in the clinic. PMID:24399995

  6. Polycomb repressive complex 2 structure with inhibitor reveals a mechanism of activation and drug resistance

    PubMed Central

    Brooun, Alexei; Gajiwala, Ketan S.; Deng, Ya-Li; Liu, Wei; Bolaños, Ben; Bingham, Patrick; He, You-Ai; Diehl, Wade; Grable, Nicole; Kung, Pei-Pei; Sutton, Scott; Maegley, Karen A.; Yu, Xiu; Stewart, Al E.

    2016-01-01

    Polycomb repressive complex 2 (PRC2) mediates gene silencing through chromatin reorganization by methylation of histone H3 lysine 27 (H3K27). Overexpression of the complex and point mutations in the individual subunits of PRC2 have been shown to contribute to tumorigenesis. Several inhibitors of the PRC2 activity have shown efficacy in EZH2-mutated lymphomas and are currently in clinical development, although the molecular basis of inhibitor recognition remains unknown. Here we report the crystal structures of the inhibitor-bound wild-type and Y641N PRC2. The structures illuminate an important role played by a stretch of 17 residues in the N-terminal region of EZH2, we call the activation loop, in the stimulation of the enzyme activity, inhibitor recognition and the potential development of the mutation-mediated drug resistance. The work presented here provides new avenues for the design and development of next-generation PRC2 inhibitors through establishment of a structure-based drug design platform. PMID:27122193

  7. Inhibition of human aromatase by myosmine.

    PubMed

    Doering, Irene L; Richter, Elmar

    2009-04-01

    Myosmine, a minor tobacco alkaloid widely occurring in food products of plant and animal origin, inhibits the conversion of testosterone to estradiol by human aromatase (IC(50): 33+/-2 microM) sevenfold more potent than nicotine (IC(50): 223+/-10 microM) and may have implications for sexual hormone homoeostasis.

  8. Orchard factors associated with resistance and cross resistance to sterol demethylation inhibitor fungicides in populations of Venturia inaequalis from Pennsylvania.

    PubMed

    Pfeufer, Emily E; Ngugi, Henry K

    2012-03-01

    Orchard management practices, such as destroying of overwintered inoculum and limiting the number of fungicide applications, are often recommended as tactics for slowing the development of resistance to sterol demethylation-inhibitor (DMI) fungicides in populations of Venturia inaequalis. However, there is little quantitative evidence relating the use of such practices to levels of resistance in orchards. The aim of this study was to evaluate the sensitivity of V. inaequalis isolates from Pennsylvania to DMI fungicides, and to identify orchard management factors related to the incidence of resistant isolates. In total, 644 single-spore V. inaequalis cultures obtained from 20 apple orchards in 2008 or 2009 were tested for sensitivity to myclobutanil, fenbuconazole, or difenoconazole. Growers provided management history of the sampled plots. Widespread shifts toward resistance to the three fungicides were noted, with mean effective concentration for 50% inhibition (EC(50)) values of 2.136, 0.786, and 0.187 μg/ml for myclobutanil, fenbuconazole, and difenoconazole, respectively. Cross resistance to the three fungicides was documented in high correlation (Spearman's r > 0.6) between mean EC(50) values for 14 orchards. Based on a 0.5-μg/ml threshold, 66 and 26% of isolates were resistant to myclobutanil and fenbuconazole, respectively, and 22% were cross resistant to the two fungicides. A significant between-year shift toward increased resistance was noted in two of three orchards surveyed in both years. Failure to use dormant copper sprays, older trees, larger orchards, orchards with ≤10 cultivars, and application of >4 DMI sprays were positively correlated (0.0001 < P < 0.05) with the incidence of resistant isolates. Isolates from orchards with >4 DMI sprays were four times as likely to be resistant to fenbuconazole (odds ratio = 4.57; P = 0.015). Isolates from orchards without dormant copper sprays were twice as likely to be cross-shifted toward resistance to all

  9. Combinations of reverse transcriptase, protease, and integrase inhibitors can be synergistic in vitro against drug-sensitive and RT inhibitor-resistant molecular clones of HIV-1.

    PubMed

    Beale, K K; Robinson, W E

    2000-06-01

    Combinations of anti-HIV agents including one or two reverse transcriptase inhibitors with a protease inhibitor are potent and effective. However, toxicities, costs and the emergence of drug-resistant organisms have compromised their long-term efficacy in people. A next, likely, target for anti-HIV therapy is HIV-1 integrase. Viral integration, catalyzed by integrase, is absolutely required for HIV replication. L-chicoric acid is a potent and selective inhibitor of HIV-1 integrase that also inhibits HIV-1 replication in cell culture. As a first step in understanding the potential role for integrase inhibitors in clinical medicine, the activities of L-chicoric acid alone and in combination with 2', 3'-dideoxycytidine, zidovudine, and a protease inhibitor, nelfinavir, were tested in vitro against molecular clones of HIV-1 resistant to reverse transcriptase inhibitors. L-chicoric acid was equally effective against a wild-type clone of HIV-1, HIV(NL4-3), or against HIV-1 resistant to either zidovudine or dideoxycytidine. L-chicoric acid was largely synergistic with zidovudine and synergistic with both dideoxycytidine and nelfinavir.

  10. Are PECTIN ESTERASE INHIBITOR Genes Involved in Mediating Resistance to Rhynchosporium commune in Barley?

    PubMed Central

    Marzin, Stephan; Hanemann, Anja; Sharma, Shailendra; Hensel, Götz; Kumlehn, Jochen; Schweizer, Günther; Röder, Marion S.

    2016-01-01

    A family of putative PECTIN ESTERASE INHIBITOR (PEI) genes, which were detected in the genomic region co-segregating with the resistance gene Rrs2 against scald caused by Rhynchosporium commune in barley, were characterized and tested for their possible involvement in mediating resistance to the pathogen by complementation and overexpression analysis. The sequences of the respective genes were derived from two BAC contigs originating from the susceptible cultivar ‘Morex’. For the genes HvPEI2, HvPEI3, HvPEI4 and HvPEI6, specific haplotypes for 18 resistant and 23 susceptible cultivars were detected after PCR-amplification and haplotype-specific CAPS-markers were developed. None of the tested candidate genes HvPEI2, HvPEI3 and HvPEI4 alone conferred a high resistance level in transgenic over-expression plants, though an improvement of the resistance level was observed especially with OE-lines for gene HvPEI4. These results do not confirm but also do not exclude an involvement of the PEI gene family in the response to the pathogen. A candidate for the resistance gene Rrs2 could not be identified yet. It is possible that Rrs2 is a PEI gene or another type of gene which has not been detected in the susceptible cultivar ‘Morex’ or the full resistance reaction requires the presence of several PEI genes. PMID:26937960

  11. Are PECTIN ESTERASE INHIBITOR Genes Involved in Mediating Resistance to Rhynchosporium commune in Barley?

    PubMed

    Marzin, Stephan; Hanemann, Anja; Sharma, Shailendra; Hensel, Götz; Kumlehn, Jochen; Schweizer, Günther; Röder, Marion S

    2016-01-01

    A family of putative PECTIN ESTERASE INHIBITOR (PEI) genes, which were detected in the genomic region co-segregating with the resistance gene Rrs2 against scald caused by Rhynchosporium commune in barley, were characterized and tested for their possible involvement in mediating resistance to the pathogen by complementation and overexpression analysis. The sequences of the respective genes were derived from two BAC contigs originating from the susceptible cultivar 'Morex'. For the genes HvPEI2, HvPEI3, HvPEI4 and HvPEI6, specific haplotypes for 18 resistant and 23 susceptible cultivars were detected after PCR-amplification and haplotype-specific CAPS-markers were developed. None of the tested candidate genes HvPEI2, HvPEI3 and HvPEI4 alone conferred a high resistance level in transgenic over-expression plants, though an improvement of the resistance level was observed especially with OE-lines for gene HvPEI4. These results do not confirm but also do not exclude an involvement of the PEI gene family in the response to the pathogen. A candidate for the resistance gene Rrs2 could not be identified yet. It is possible that Rrs2 is a PEI gene or another type of gene which has not been detected in the susceptible cultivar 'Morex' or the full resistance reaction requires the presence of several PEI genes.

  12. The plant polyphenol butein inhibits testosterone-induced proliferation in breast cancer cells expressing aromatase.

    PubMed

    Wang, Yun; Chan, Franky L; Chen, Shiuan; Leung, Lai K

    2005-05-20

    Chalcones are precursor compounds for flavonoid synthesis in plants, and they can also be synthesized in laboratory. Previous study has documented some of the pharmacological applications of these compounds. Estrogen has long been associated with the initiation and promotion of breast cancer. Inhibiting estrogen synthesis can be effective in the prevention and treatment of the disease. Since most breast cancers received estrogen supplied from local tissues, we employed a breast cancer cell line expressing aromatase to screen for the inhibitory potentials of five hydroxychalcones, i.e. 2-hydroxychalcone, 2'-hydroxychalcone, 4-hydroxychalcone, 4,2',4'-trihydroxy-chalcone (isoquiritigenin), 3,4,2',4'-tetrahydroxychalcone (butein). In the preliminary results, butein was found to be the strongest inhibitor among the tested compounds, and its IC(50) value was 3.75 microM. Subsequent enzyme kinetic study revealed that butein acted on aromatase with a mixed type of inhibition and the K(i) value was determined to be 0.32 microM. Cell proliferation assay indicated that the cell number increased by 10 nM-testosterone treatment was significantly reduced by 5 microM butein, and the administration of flutamide could not reverse the effect. The present study illustrated that butein was an aromatase inhibitor and a potential natural alternative for the chemoprevention or therapy of breast cancer.

  13. Synthesis of Casimiroin and Optimization of Its Quinone Reductase 2 and Aromatase Inhibitory Activities

    SciTech Connect

    Maiti, Arup; Reddy, P.V. Narasimha; Sturdy, Megan; Marler, Laura; Pegan, Scott D.; Mesecar, Andrew D.; Pezzuto, John M.; Cushman, Mark

    2009-08-07

    An efficient method has been developed to synthesize casimiroin (1), a component of the edible fruit of Casimiroa edulis, on a multigram scale in good overall yield. The route was versatile enough to provide an array of compound 1 analogues that were evaluated as QR2 and aromatase inhibitors. In addition, X-ray crystallography studies of QR2 in complex with compound 1 and one of its more potent analogues has provided insight into the mechanism of action of this new series of QR2 inhibitors. The initial biological investigations suggest that compound 1 and its analogues merit further investigation as potential chemopreventive or chemotherapeutic agents.

  14. Synthesis of Casimiroin and Optimization of Its Quinone Reductase 2 and Aromatase Inhibitory Activities

    PubMed Central

    Maiti, Arup; Narasimha Reddy, P. V.; Sturdy, Megan; Marler, Laura; Pegan, Scott D.; Mesecar, Andrew D.; Pezzuto, John M.; Cushman, Mark

    2009-01-01

    An efficient method has been developed to synthesize casimiroin (1), a component of the edible fruit of Casimiroa edulis, on a multigram scale in good overall yield. The route was versatile enough to provide an array of compound 1 analogues that were evaluated as QR2 and aromatase inhibitors. In addition, X-ray crystallography studies of QR2 in complex with compound 1 and one of its more potent analogs has provided insight into the mechanism of action of this new series of QR2 inhibitors. The initial biological investigations suggest that compound 1 and its analogues merit further investigation as potential chemopreventive or chemotherapeutic agents. PMID:19265439

  15. Genistein induces breast cancer-associated aromatase and stimulates estrogen-dependent tumor cell growth in in vitro breast cancer model.

    PubMed

    van Duursen, M B M; Nijmeijer, S M; de Morree, E S; de Jong, P Chr; van den Berg, M

    2011-11-18

    In breast cancer, the interaction between estrogen-producing breast adipose fibroblasts (BAFs) and estrogen-dependent epithelial tumor cells is pivotal. Local estrogen production is catalyzed by aromatase, which is differentially regulated in disease-free and tumorigenic breast tissue. The use of aromatase inhibitors to block local estrogen production has proven effective in treatment of estrogen-dependent breast cancer. However, a major problem during breast cancer treatment is the sudden onset of menopause and many women seek for alternative medicines, such as the soy isoflavone genistein. In this study, we show that genistein can induce estrogen-dependent MCF-7 tumor cell growth and increase breast cancer-associated aromatase expression and activity in vitro. We have previously developed an in vitro breast cancer model where the positive feedback loop between primary BAFs and estrogen-dependent MCF-7 tumor cells is operational, thereby representing a more natural in vitro model for breast cancer. In this model, genistein could negate the growth inhibitory action of the aromatase inhibitor fadrozole at physiologically relevant concentrations. These data suggest that soy-based supplements might affect the efficacy of breast cancer treatment with aromatase inhibitors. Considering the high number of breast cancer patients using soy supplements to treat menopausal symptoms, the increasing risk for adverse interactions with breast cancer treatment is of major concern and should be considered with care.

  16. High-content screening identifies kinase inhibitors that overcome venetoclax resistance in activated CLL cells

    PubMed Central

    Oppermann, Sina; Ylanko, Jarkko; Shi, Yonghong; Hariharan, Santosh; Oakes, Christopher C.; Brauer, Patrick M.; Zúñiga-Pflücker, Juan C.; Leber, Brian; Spaner, David E.

    2016-01-01

    Novel agents such as the Bcl-2 inhibitor venetoclax (ABT-199) are changing treatment paradigms for chronic lymphocytic leukemia (CLL) but important problems remain. Although some patients exhibit deep and durable responses to venetoclax as a single agent, other patients harbor subpopulations of resistant leukemia cells that mediate disease recurrence. One hypothesis for the origin of resistance to venetoclax is by kinase-mediated survival signals encountered in proliferation centers that may be unique for individual patients. An in vitro microenvironment model was developed with primary CLL cells that could be incorporated into an automated high-content microscopy-based screen of kinase inhibitors (KIs) to identify agents that may improve venetoclax therapy in a personalized manner. Marked interpatient variability was noted for which KIs were effective; nevertheless, sunitinib was identified as the most common clinically available KI effective in overcoming venetoclax resistance. Examination of the underlying mechanisms indicated that venetoclax resistance may be induced by microenvironmental signals that upregulate antiapoptotic Bcl-xl, Mcl-1, and A1, which can be counteracted more efficiently by sunitinib than by ibrutinib or idelalisib. Although patient-specific drug responses are common, for many patients, combination therapy with sunitinib may significantly improve the therapeutic efficacy of venetoclax. PMID:27297795

  17. Structure-guided optimization of protein kinase inhibitors reverses aminoglycoside antibiotic resistance

    PubMed Central

    Stogios, Peter J.; Spanogiannopoulos, Peter; Evdokimova, Elena; Egorova, Olga; Shakya, Tushar; Todorovic, Nick; Capretta, Alfredo; Wright, Gerard D.; Savchenko, Alexei

    2013-01-01

    SYNOPSIS Activity of the aminoglycoside phosphotransferase APH(3’)-Ia leads to resistance to aminoglycoside antibiotics in pathogenic Gram-negative bacteria, and contributes to the clinical obsolescence of this class of antibiotics. One strategy to rescue compromised antibiotics such as aminoglycosides is targeting the enzymes that confer resistance with small molecules. Previously we demonstrated that eukaryotic protein kinase (ePK) inhibitors could inhibit APH enzymes, due to the structural similarity between these two enzyme families. However, limited structural information of enzyme-inhibitor complexes hindered interpretation of the results. As well, cross-reactivity of compounds between APHs and ePKs represents an obstacle to their use as aminoglycoside adjuvants to rescue aminoglycoside antibiotic activity. Here, we structurally and functionally characterize inhibition of APH(3’)-Ia by three diverse chemical scaffolds – anthrapyrazolone, 4-anilinoquinazoline and pyrazolopyrimidine (PP) – and reveal distinctions in the binding mode of anthrapyrazolone and PP compounds to APH(3’)-Ia versus ePKs. Using this observation, we identify PP-derivatives that select against ePKs, attenuate APH(3’)-Ia activity and rescue aminoglycoside antibiotic activity against a resistant E. coli strain. The structures presented here and these inhibition studies provide an important opportunity for structure-based design of compounds to target aminoglycoside phosphotransferases for inhibition, potentially overcoming this form of antibiotic resistance. PMID:23758273

  18. High-content screening identifies kinase inhibitors that overcome venetoclax resistance in activated CLL cells.

    PubMed

    Oppermann, Sina; Ylanko, Jarkko; Shi, Yonghong; Hariharan, Santosh; Oakes, Christopher C; Brauer, Patrick M; Zúñiga-Pflücker, Juan C; Leber, Brian; Spaner, David E; Andrews, David W

    2016-08-18

    Novel agents such as the Bcl-2 inhibitor venetoclax (ABT-199) are changing treatment paradigms for chronic lymphocytic leukemia (CLL) but important problems remain. Although some patients exhibit deep and durable responses to venetoclax as a single agent, other patients harbor subpopulations of resistant leukemia cells that mediate disease recurrence. One hypothesis for the origin of resistance to venetoclax is by kinase-mediated survival signals encountered in proliferation centers that may be unique for individual patients. An in vitro microenvironment model was developed with primary CLL cells that could be incorporated into an automated high-content microscopy-based screen of kinase inhibitors (KIs) to identify agents that may improve venetoclax therapy in a personalized manner. Marked interpatient variability was noted for which KIs were effective; nevertheless, sunitinib was identified as the most common clinically available KI effective in overcoming venetoclax resistance. Examination of the underlying mechanisms indicated that venetoclax resistance may be induced by microenvironmental signals that upregulate antiapoptotic Bcl-xl, Mcl-1, and A1, which can be counteracted more efficiently by sunitinib than by ibrutinib or idelalisib. Although patient-specific drug responses are common, for many patients, combination therapy with sunitinib may significantly improve the therapeutic efficacy of venetoclax.

  19. High Affinity Inha Inhibitors with Activity Against Drug-Resistant Strains of Mycobacterium Tuberculosis

    SciTech Connect

    Sullivan,T.; Truglio, J.; Boyne, M.; Novichenok, P.; Zhang, X.; Stratton, C.; Li, H.; Kaur, T.; Amin, A.; et al.

    2006-01-01

    Novel chemotherapeutics for treating multidrug-resistant (MDR) strains of Mycobacterium tuberculosis (MTB) are required to combat the spread of tuberculosis, a disease that kills more than 2 million people annually. Using structure-based drug design, we have developed a series of alkyl diphenyl ethers that are uncompetitive inhibitors of InhA, the enoyl reductase enzyme in the MTB fatty acid biosynthesis pathway. The most potent compound has a Ki{prime} value of 1 nM for InhA and MIC{sub 99} values of 2-3 {micro}g mL{sup -1} (6-10 {micro}M) for both drug-sensitive and drug-resistant strains of MTB. Overexpression of InhA in MTB results in a 9-12-fold increase in MIC{sub 99}, consistent with the belief that these compounds target InhA within the cell. In addition, transcriptional response studies reveal that the alkyl diphenyl ethers fail to upregulate a putative efflux pump and aromatic dioxygenase, detoxification mechanisms that are triggered by the lead compound triclosan. These diphenyl ether-based InhA inhibitors do not require activation by the mycobacterial KatG enzyme, thereby circumventing the normal mechanism of resistance to the front line drug isoniazid (INH) and thus accounting for their activity against INH-resistant strains of MTB.

  20. Structure-guided optimization of protein kinase inhibitors reverses aminoglycoside antibiotic resistance.

    PubMed

    Stogios, Peter J; Spanogiannopoulos, Peter; Evdokimova, Elena; Egorova, Olga; Shakya, Tushar; Todorovic, Nick; Capretta, Alfredo; Wright, Gerard D; Savchenko, Alexei

    2013-09-01

    Activity of the aminoglycoside phosphotransferase APH(3')-Ia leads to resistance to aminoglycoside antibiotics in pathogenic Gram-negative bacteria, and contributes to the clinical obsolescence of this class of antibiotics. One strategy to rescue compromised antibiotics such as aminoglycosides is targeting the enzymes that confer resistance with small molecules. We demonstrated previously that ePK (eukaryotic protein kinase) inhibitors could inhibit APH enzymes, owing to the structural similarity between these two enzyme families. However, limited structural information of enzyme-inhibitor complexes hindered interpretation of the results. In addition, cross-reactivity of compounds between APHs and ePKs represents an obstacle to their use as aminoglycoside adjuvants to rescue aminoglycoside antibiotic activity. In the present study, we structurally and functionally characterize inhibition of APH(3')-Ia by three diverse chemical scaffolds, anthrapyrazolone, 4-anilinoquinazoline and PP (pyrazolopyrimidine), and reveal distinctions in the binding mode of anthrapyrazolone and PP compounds to APH(3')-Ia compared with ePKs. Using this observation, we identify PP derivatives that select against ePKs, attenuate APH(3')-Ia activity and rescue aminoglycoside antibiotic activity against a resistant Escherichia coli strain. The structures described in the present paper and the inhibition studies provide an important opportunity for structure-based design of compounds to target aminoglycoside phosphotransferases for inhibition, potentially overcoming this form of antibiotic resistance.

  1. Identification of Long Non-Coding RNAs Deregulated in Multiple Myeloma Cells Resistant to Proteasome Inhibitors

    PubMed Central

    Malek, Ehsan; Kim, Byung-Gyu; Driscoll, James J.

    2016-01-01

    While the clinical benefit of proteasome inhibitors (PIs) for multiple myeloma (MM) treatment remains unchallenged, dose-limiting toxicities and the inevitable emergence of drug resistance limit their long-term utility. Disease eradication is compromised by drug resistance that is either present de novo or therapy-induced, which accounts for the majority of tumor relapses and MM-related