用于相对和绝对定量的等量异位标签-二维液相色谱-串联质谱法分析雌二醇和血清剥夺对乳腺癌细胞内蛋白质含量的影响

雌激素和血清中的激素及各种生长因子在乳腺癌的发生、发展过程中发挥着重要的作用。研究雌激素和血清对乳腺癌细胞中蛋白质组成和含量的影响对于阐明雌激素和血清对乳腺癌细胞影响的分子机理具有重要的意义。利用四重用于相对和绝对定量的等量异位标签(isobaric tags for relative and absolute quantification, iTRAQ)标记结合二维液相色谱-串联质谱(two-dimensional liquid chromatography-tandem mass spectrometry, 2D-LC-MS/MS)对雌二醇(17β-oestradiol, E2)和血清各自以及共同作用对MCF7乳腺癌细胞内蛋白质表达的影响进行了比较,共鉴定到置信度在95%以上的蛋白质576种,其中各组相对于正常培养组的细胞共找到26种差异在1倍以上的蛋白质,其中10种上调,16种下调。研究发现E2和血清可显著影响细胞内与蛋白质合成相关的蛋白质的水平。实验结果表明: iTRAQ-LC-MS/MS是进行多个样品差异蛋白组比较的一种有效方法。     

3β-羟基雄甾-4-烯-6,17-二酮的合成

去氢表雄酮醋酸酯经过氧甲酸环氧化、高碘酸开环、IBX氧化、脱水和碱性水解等5步反应,以55%的总收率合成得到了非雄性激素芳香化酶抑制剂3β-羟基雄甾-4-烯-6,17-二酮。在环氧化反应中,利用价廉、易制备的过氧甲酸以几乎定量的收率得到了环氧化合物。使用IBX氧化邻二醇,以98.5%的收率得到氧化产物,避免了使用处理困难并且污染环境的铬试剂。     

人胎盘的生殖内分泌学研究——Ⅱ.细胞滋养层细胞的激素分泌功能

本文采用了无血清培养技术,研究了细胞滋养层细胞在体外分泌hCG、孕酮、雌激素、cGnRH和β-内啡肽的功能,在8天的培养中发现,细胞滋养层细胞在培养24h去除内源激素后能持续地分泌少量的hCG;与此不同,孕酮的分泌量逐日上升,第6天的孕酮分泌量为第1天的两倍;在外加雌激素的前身物雄烯二酮的情况下,细胞能合成雌激素,其产量亦是逐日上升,反映出细胞芳香化酶活性的不断提高,这种酶活性的提高与雄烯二酮的存在与否无关;在两种滋养层细胞培养中,唯有细胞滋养层细胞培养液中测得有cGnRH,产量以第2天为最高,接近4pg/10~5个细胞,合体滋养层细胞在离体下能分泌β-内啡肽,但其产量逐日下降,与此相反,细胞滋养层细胞的培养液中不但测到β-内啡肽的存在而且其含量在培养4—6天间明显上升。     

基于刚性共轭聚苯类的树枝型β－二酮的合成

本文应用简单的合成方法，利用1-溴-3,5-二碘苯作为新颖的重复单元合成了一类高世代的聚苯类树枝型β－二酮并对其进行了详细的表征。研究了这类树枝型β－二酮在不同溶剂中的吸收和发射光谱，表明存在着不太明显的溶剂效应。同时，也对这类树枝型β－二酮的热性质进行了研究。     

4-取代环芬尼类化合物的合成及标记

以Cyclofenil为原料, 合成得到一系列氟乙氧基衍生物. 通过受体结合实验, 筛选得到一个对雌激素受体(ER)具有高特异性和高相对亲和力(RBA)的化合物Penta-Cyclofenil-FEt. 通过实验的优化设计, 合成得到相应的氟标记前体, 产物通过IR, UV, ¹H NMR, ¹⁹F NMR或ESI-HRMS进行了表征. 在冷试条件下, 得到了冷氟取代参比化合物. 在热试验条件下, 得到F-18标记产物. 此类非甾体雌激素受体分子探针的合成, 为PET (positron emission tomography)显像雌激素受体β从而评估乳腺癌的恶化程度奠定了基础.     

灰化前处理-电感耦合等离子体原子发射光谱法测定阿那曲唑中钯的残留量

阿那曲唑商品名为瑞婷、瑞宁得,为强效非甾体类芳香化酶抑制剂,可降低血浆雌激素水平,产生抑制乳腺肿瘤生长的作用,临床上广泛用于绝经后妇女的晚期乳腺癌治疗[1-2]。由于阿那曲唑在制备过程中要用到钯作催化剂,其残留会对人体健康带来一定的危害,所以必须对钯的残留量做准确的测定[3]。微量钯的测定多采用分光光度法[4]或原子吸收光谱法[5-6],也有关于电感耦合等离子体原子发射     

2-羟基脱氧雌酚酮的合成

2-羟基雌甾化合物是人体内对生理机能有重要意义的雌激素代谢产物,其在生物体内的含量极少,需用化学方法合成来满足研究、应用的需要。迄今为止,已报道了较多合成2-羟基和4-羟基雌甾化合物的方法[1-3],但选择性和产率并不十分理想。我们在文献报道的合成路线[1]的基础上,合成了     

1,5-二芳基-3-(2-羟基-4,6-二甲氧基苯基)-2-吡唑啉的合成及铜离子荧光探针行为

设计合成了4个1,5-二芳基-3-(2-羟基-4,6-二甲氧基苯基)-2-吡唑啉化合物(4a～4d).其结构由IR,1HNMR,MS和元素分析确认.通过紫外光谱和荧光光谱研究了化合物对铜离子的选择性识别作用,结果发现,化合物4a～4d均可以选择性地识别铜离子,作为铜离子荧光探针,受常见离子干扰较小,选择性较高.     

1-乙酰基-3-(2-羟基-4,6-二甲氧基苯基)-5-苯基-2-吡唑啉的合成及锌离子探针的研究

设计合成了1-乙酰基-3-(2-羟基-4,6-二甲氧基苯基)-5-苯基-2-吡唑啉(4), 测试了其紫外光谱和荧光光谱, 研究了其对锌离子的选择性识别作用. 结果表明, 化合物4作为锌离子荧光探针, 受常见离子的干扰较小, 对于锌离子有着较高的选择性和较低的检出限.     

过氧化物酶新型荧光底物3,4-二氢-2(1H)-喹喔啉酮的反应机理及性能的研究

合成了过氧化物酶新型荧光底物3,4-二氢-2(1H)-喹喔啉酮(DHQXL). 在过氧化物酶催化下,该化合物可被H₂O₂氧化生成强荧光物质2(1H)－喹喔啉酮(QXL). 用高压液相色谱法分离得荧光产物纯品,经核磁共振谱和质谱证实其结构,确证了提出的酶催化反应机理. 对该化合物作为荧光底物的性能研究表明,它是一个很有应用前景的过氧化物酶的新型荧光底物.     

Development and validation of a liquid chromatography–tandem mass spectrometry method for the simultaneous quantification of tamoxifen, anastrozole, and letrozole in human plasma and its application to a clinical study

There is substantial evidence that circulating estrogens promote the proliferation of breast cancer. Consequently, adjuvant hormonal treatment strategies targeting estrogen action have been established. Such hormonal therapies include selective estrogen receptor modulators, such as tamoxifen, which interfere at the estrogen receptors directly, or non-steroidal aromatase inhibitors, such as anastrozole and letrozole, which inhibit estrogen synthesis through blocking the aromatase, a key enzyme of estrogen production. Despite considerable therapeutic success, in several cases, the use of these drugs is limited by side effects that have been described to significantly impair the adherence of patients to endocrine treatment. However, objective data concerning patient adherence and its clinical relevance are limited. One promising approach to check patient-reported adherence is drug monitoring in human plasma. Therefore, a liquid chromatography–tandem mass spectrometry method to determine the plasma concentrations of tamoxifen, anastrozole, and letrozole has been developed and fully validated according to guidelines for clinical and forensic toxicology. The validation criteria evaluated were selectivity, linearity, accuracy and precision, limit of quantification, recovery and matrix effects, sample stability, and carryover. The six-point calibration curves showed linearity over the range of concentrations from 25 to 500 ng/ml for tamoxifen, 5 to 200 ng/ml for anastrozole, and 10 to 300 ng/ml for letrozole. The intra- and inter-day precision and accuracies were always better than 15%. The validated procedure was successfully applied to a clinical study (Patient-Reported Outcomes in Breast Cancer Patients undergoing Endocrine Therapy, PRO-BETh). A major aim of PRO-BETh study is the comprehensive evaluation of adherence to treatment in pre- and post-menopausal women with breast cancer. Plasma samples of 310 breast cancer patients undergoing anti-estrogen therapy were analyzed. Eight samples did not contain a quantifiable amount of drug, strongly indicating non-adherence of the corresponding patients to adjuvant breast cancer treatment. Furthermore, plasma concentrations at the lower end of the observed plasma level distribution might represent a hint but not a confirmation for non-adherence in terms of non-daily and irregular intake of the prescribed drug.     

Reconsidering Aromatase for Breast Cancer Treatment: New Roles for an Old Target

The current therapeutic approach for the treatment of hormone dependent breast cancer includes interference with estrogen receptors via either selective modulators or estrogens deprivation, by preventing their biosynthesis with aromatase inhibitors. Severe side effects and acquired resistance are drawbacks of both drug classes, and the efforts to overcome these issues still allow for research in this field to be animated. This review reports on recent findings that have opened new avenues for reconsidering the role of aromatase enzymes (and estrogen receptors) leading to the possibility of looking at well-known targets in a new perspective.     

Design and pharmacophoric identification of flavonoid scaffold-based aromatase inhibitors

Aromatase is a crucial enzyme for the catalysis of aromatization reaction at the last and rate-limiting step involved in the conversion of androgenic substrates to an estrogenic substrate. A hormone-dependent breast cancer in postmenopausal woman can be cured by inhibition of estrogen biosynthesis by the help of aromatase inhibitors (AIs). The mode of interactions of flavonones with the active site of aromatase has been studied in search of potent and selective AIs as a substitute of the natural steroidal ligand. Structure-based computational approach namely, molecular docking simulations were performed to investigate the structural features of the docked complex of aromatase and flavonoid ligands. A nonsteroidal flavonoid pharmacophore showing electrostatic and steric features for selective binding within the main pocket of the catalytic active site of aromatase has been identified as an outcome of the study. The binding affinity of quercetin and isoflavone were predicted within aromatase. Isoflavone was used as a negative control to compare its binding affinities with the selected dataset. The predicted binding affinity of negative control isoflavone was in accordance with its in vitro AI efficacy. Isoflavone showed poor binding affinity and ranked last in terms of MolDock score (−86.309 kcal/molÅ) compared to dataset molecules. The generated pharmacophoric information will be helpful for the synthetic chemist to design and synthesize selective AIs with comparable binding affinity to the natural steroidal ligand.     

Identification of the aromatase inhibitor letrozole in urine by gas chromatography/mass spectrometry

Letrozole (1-(bis-(4-cyanophenyl)methyl)-1,2,4-triazole) is used therapeutically as a non-steroidal aromatase inhibitor (Femara) to treat hormone-sensitive breast cancer in postmenopausal women. For doping purposes it may be used to counteract the adverse effects of an extensive abuse of anabolic androgenic steroids (gynaecomastia) and to increase the testosterone concentration by stimulation of the testosterone biosynthesis. The use of aromatase inhibitors has been prohibited by IOC/WADA regulations for male and female athletes since September 2001 and January 2005, respectively. Spot urine samples from women suffering from metastatic breast cancer and being treated with letrozole were collected and analysed to develop/optimise the detection system for metabolites of letrozole to allow the identification of athletes who do not comply with the internationally prohibited use of this cancer drug. The assay was based on gas chromatography/mass spectrometry (GC/MS) and the main metabolite of letrozole (bis-4-cyanophenylmethanol) was identified by comparison of its mass spectrum and retention time with that of a bis-4-cyanophenylmethanol reference. The full-scan spectrum, diagnostic ions and a validation of the method for the analysis of bis-4-cyanophenylmethanol are presented.     

Computational design and experimental discovery of an antiestrogenic peptide derived from alpha-fetoprotein

Breast cancer is the most common cancer among women, and tamoxifen is the preferred drug for estrogen receptor-positive breast cancer treatment. Many of these cancers are intrinsically resistant to tamoxifen or acquire resistance during treatment. Consequently, there is an ongoing need for breast cancer drugs that have different molecular targets. Previous work has shown that 8-mer and cyclic 9-mer peptides inhibit breast cancer in mouse and rat models, interacting with an unsolved receptor, while peptides smaller than eight amino acids did not. We show that the use of replica exchange molecular dynamics predicts the structure and dynamics of active peptides, leading to the discovery of smaller peptides with full biological activity. Simulations identified smaller peptide analogues with the same conserved reverse turn demonstrated in the larger peptides. These analogues were synthesized and shown to inhibit estrogen-dependent cell growth in a mouse uterine growth assay, a test showing reliable correlation with human breast cancer inhibition.     

Quantification of tamoxifen and metabolites and soy isoflavones in human plasma using liquid chromatography with electrospray ionization tandem mass spectrometry

Tamoxifen is an important estrogen receptor antagonist used successfully for the treatment and prevention of breast cancer. The use of complementary and alternative medicines is an increasingly popular means for patients to participate in their own health care, and soy products, which contain phytoestrogens, have been widely promoted for possible beneficial effects on menopausal symptoms. The possibility that soy isoflavones could reduce tamoxifen efficacy has been demonstrated in animal models of post-menopausal breast cancer, but the occurrence of such an effect in women has not been explored. This paper describes the development and validation of a sensitive method using solid-phase extraction and isotope dilution liquid chromatography/tandem mass spectrometry with multiple reaction monitoring for the concurrent analysis of the major soy isoflavones (genistein and daidzein), an important metabolite of daidzein (equol), tamoxifen, and its important metabolites (4-hydroxytamoxifen, N-desmethyltamoxifen, and 4-hydroxy-N-desmethyltamoxifen or "endoxifen") in the serum of rats and women. The limits of quantification achieved are sufficient to determine accurately and precisely the concentrations of all of these analytes in women consuming soy foods and/or therapeutic doses of tamoxifen at levels consistent with modulation of estrogen receptor-mediated functions. These procedures enable future investigations of the possible impact of diet on the outcome of breast cancer therapy.     

Comparative profiling of plasma proteome from breast cancer patients reveals thrombospondin-1 and BRWD3 as serological biomarkers

Breast cancer is the most common cancer in women worldwide. It is necessary to identify biomarkers for early detection, to make accurate prognoses, and to monitor for any recurrence of the cancer. In order to identify potential breast cancer biomarkers, we analyzed the plasma samples of women diagnosed with breast cancer and age-matched normal healthy women by mTRAQ-based stable isotope-labeling mass spectrometry. We identified and quantified 204 proteins including thrombospondin-1 (THBS1) and bromodomain and WD repeat-containing protein 3 (BRWD3) which were increased by more than 5-fold in breast cancer plasma. The plasma levels of the two proteins were evaluated by Western blot assay to confirm for their diagnostic value as serum markers. A 1.8-fold increase in BRWD3 was observed while comparing the plasma levels of breast cancer patients (n = 54) with age-matched normal healthy controls (n = 30), and the area under the receiver operating characteristic curve (AUC) was 0.917. THBS1 was detected in pooled breast cancer plasma at the ratio similar to mTRAQ ratio (> 5-fold). The AUC value for THBS1 was 0.875. The increase of THBS1 was more prominent in estrogen receptor negative and progesterone receptor negative patients than receptor-positive patients. Our results are evidence of the diagnostic value of THBS1 in detecting breast cancer. Based on our findings, we suggest a proteomic method for protein identification and quantification lead to effective biomarker discovery.     

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

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.     

Design,synthesis and biological evaluation of 1,4-dihydrothieno[3′,2′:5,6]thiopyrano[4,3-c]pyrazole-3-carboxylic amide derivatives as potential estrogen receptor antagonists

The estrogen receptor is a target for therapeutic agents for hormone replacement in menopausal women,osteoporosis, reproductive cancers such as breast cancer,uterine cancer and prostate cancer.1,4-Dihydrothieno[3’,2’:5,6]thiopyrano[4,3- c]pyrazole-3-carboxylic amide derivatives were designed,synthesized and biological evaluated as potential estrogen receptor antagonists.     

Chemistry and Molecular Biology in the Search for New LHRH Antagonists

Hormones—and in particular, the sex hormones—were the first growth factors discovered to be involuntary helpers of cancer. Female breast cancer and male prostate cancer are the best known examples of tumors acknowledged to be hormone-dependent. A look at cancer statistics shows that breast cancer is still the most frequent cancer in women; in men, prostate cancer plays a similarly dominant role with increasing age. Shutting down the main production site of the sex hormones estrogen and testosterone either by removing the ovaries or by castration is a well-known and often effective therapy; however, these procedures can be problematic due to the concomittant psychological stress. Modern hormone therapy for advanced breast cancer and prostate cancer attempts to spare the patient such irreversible operative procedures for as long as possible, by using hormone antagonists, such as the LHRH antagonists, which hinder deployment of the hormone itself and thus its growth-promoting activity.