新型雌激素受体ER-α36与乳腺癌

雌激素受体(estrogen receptor,ER)是诊断和治疗乳腺癌的分子标志和靶点.雌激素受体包括ER-α和ER-β,其中ER-α有ER-α66、ER-α46和ER-α36三种亚型.ER-α36作为新型雌激素受体,参与膜起始的雌激素信号或非基因组雌激素信号转导,在肿瘤细胞的增殖、分化、侵袭和转移等过程中发挥作用.胃癌、子宫内膜腺癌、前列腺癌、尤其是乳腺癌的发生发展与ER-α36密切相关.本文介绍了ER-α36的结构域特点,ER-α36介导的信号通路及ER-α36在乳腺癌治疗中的作用研究进展.     

Estrogen-independent effects of ER-α36 in ER-negative breast cancer

Estrogen receptor-alpha 36 (ER-α36) is a variant of ER-α that has been found to be expressed in conventional ER (ER-α66)-negative breast cancer cell lines and human breast cancer samples. In this study, we found that, using immunohistochemical study, ER-α36 expression was significantly higher in ER-negative tumors than in ER-positive tumors although the expression was not associated with other clinicopathological characteristics. We then constructed an ER-α36-specific microRNA hairpin vector and established stable ER-α36 knockdown cells, and found that the knockdown cells were more sensitive to paclitaxel; the c-Jun N-terminal kinase pathway appeared to be involved in the mechanism. Downregulation of ER-α36 also resulted in decreased migration and invasion. These changes were estrogen independent. Our findings indicated that target ER-α36 may be a strategy for treating ER-negative breast cancers.     

ER-α36在妇科肿瘤中介导非基因组效应研究进展

雌激素参与调节女性多种生殖或非生殖组织的正常生长、分化和运行。外源雌激素(xenoestrogen,XEs)的介入干扰机体内正常的生理平衡,并在具有雌激素依赖性细胞中通过雌激素受体(estrogen receptor,ER)介导基因组和非基因组途径发挥雌激素效应。然而,膜雌激素受体,特别是ER-α36介导XEs产生的非基因组途径因其低剂量、低亲和力、非线性、快速信号转导、整合等效应而备受关注。在XEs的刺激下,ER-α36可通过非基因组途径激活cAMP、PKC、Ca~(2+)、MAPK/ERK、PI3K/Akt等下游通路诱导产生雌激素效应。研究ER-α36和肿瘤细胞的相关性对肿瘤的发生和治疗具有重要意义。重点介绍ER-α36在子宫内膜癌、乳腺癌和卵巢癌等妇科肿瘤中介导非基因组产生的雌激素效应及其在疾病治疗过程中产生的影响,以期为妇科肿瘤预防和治疗提供一定的理论指导。     

雌激素膜受体ER-α36及其在人脑胶质瘤中的作用

脑胶质瘤是最为常见的颅内恶性肿瘤,表现为浸润性生长,对放化疗的敏感性低,复发率高,发病具有男女差异,但是雌激素受体的表达并没有明显的性别差异等特点,目前尚缺乏有效的应对策略。2005年首次发现并克隆的新型雌激素受体ER-α36,在多种男女高发恶性肿瘤细胞中高表达,并通过激活MAPK、PI3K/AKT非基因组雌激素信号通路刺激细胞恶性增殖,并能同时介导雌激素和抗雌激素的促生长作用。本文综述ER-α36与脑胶质瘤患者发生发展的相关性,也为临床以ER-α36为靶点治疗恶性胶质瘤提供新思路和科学依据。     

雌激素受体-α36 (ER-α36)介导乳腺癌细胞的顺铂耐药性

雌激素受体-α36 (estrogen receptor-α36, ER-α36)在乳腺癌细胞中对顺铂耐药性的作用和机制尚不清楚。本研究考察了ER-α36在乳腺癌细胞中的表达及ER-α36对顺铂耐药性的影响和机制。Western blotting分析显示,顺铂诱导人乳腺癌细胞MCF-7中ER-α36的上调。细胞计数8试剂盒(cell counting kit-8,CCK-8)和细胞集落形成实验显示,过表达ER-α36显著提高了MCF-7细胞的增殖能力和集落形成能力,而敲低ER-α36则可抑制MCF-7细胞的增殖能力和集落形成能力。5μg/mL顺铂处理可激活EGFR/HER-2/ERK信号。敲低ER-α36可显著抑制MCF-7/DDP或MCF-7/ER-α36细胞中EGFR/HER-2/ERK信号的激活。抑制EGFR/HER-2/ERK信号可降低MCF-7/ER-α36细胞的增殖能力。总之,本研究证明ER-α36的上调通过激活EGFR/HER-2/ERK信号提高了乳腺癌细胞的顺铂耐药性。靶向ER-α36可能是提高顺铂敏感性的有效策略。     

Biosynthesis of 1α,2α,3β-trihydroxy-p-menthane by Fusicoccum amygdali

Measurement of isotope ratios in 1α,2α,3β-trihydroxy-p-menthane, which has been biosynthesized in Fusicoccum amygdali from ³H- and ¹⁴C-labelled mevalonate and in its degradation product diosphenol indicates that: (a) four tritium atoms arising from [5-³H₂, 2-¹⁴C]MVA are retained, one more than suggested from the hydroxylation pattern, (b) menth-2-ene-1-ol is generated from an α-terpinyl cation through a 1,3-hydride shift and (c) trans-cleavage of an α-epoxide by hydrolysis gives 1α,2α,3β-trihydroxy-p-menthane.     

Advances in the understanding of the structure and function of ER-α36,a novel variant of human estrogen receptor-alpha

Estrogen receptors (ERs) belong to the nuclear receptor superfamily, whose members include ER-α66, ER-α36, ER-α46 and ER-β. Each receptor performs specific functions through binding with a specific ligand, such as estrogen. Recently, ER-α36, a novel variant of human estrogen receptor-alpha (ER-α), was identified and cloned. ER-α36 inhibits, in a dominant-negative manner, the transactivation of both the wild-type ER-α (ER-α66) and ER-β. As a predominantly membrane-based ER, ER-α36 mediates nongenomic estrogen signaling and is involved in the resistance of breast cancer to endocrine therapy, i.e., tamoxifen. This review summarizes recent studies on the structure and function of ER-α36 and the relationship of ER-α36 with cancer, with special emphasis on its function in the resistance of breast cancer to endocrine therapy.     

Differential expression of VEGF-A mRNA by 17β-estradiol in breast tumor cells lacking classical ER-α may be mediated through a variant form of ER-α

Beta-estradiol (17beta-E2) augments VEGF-A expression in various estrogen targeted organs and cells including breast tumor derived cell lines, via an ER-alpha mediated pathway. Ironically, 17beta-E2 is able to regulate some genes via ER-alpha independent pathways. In the present study, we sought to determine whether 17beta-E2 can modulate VEGF-A expression in absence of ER-alpha, and therefore, three different cell lines including ER-alpha+ MCF-7, and ER-alpha SKBR-3 and HMEC were used for this study. The present study demonstrates that 17beta-E2 also induces VEGF-A mRNA expression in ER-negative SKBR-3 breast tumor cells in a manner similar to that observed in ER-positive MCF-7 cells. Blocking the induced-expression by antiestrogen ICI 182,780 indicates the induction pathway is ER dependent. While ER-alpha mRNA is absent in both HMEC and SKBR-3 cells, the impact of estrogen was found only in SKBR-3 cells, suggesting the existence of an analogue to ER-alpha or overlapping signal in these cells. Consistent with this suggestion, the present studies demonstrate the existence of an ER-alpha(var2) protein in MCF-7 and in SKBR-3 cells. This variant is predominantly localized in the nuclei of SKBR-3 cells. Importantly, specific binding of 17beta-E2 by these cells suggest the ER-alpha(var2) may act as active receptor in SKBR-3 cells.     

Disruption of the ER-α36-EGFR/HER2 Positive Regulatory Loops Restores Tamoxifen Sensitivity in Tamoxifen Resistance Breast Cancer Cells

Tamoxifen provided a successful treatment for ER-positive breast cancer for many years. However, most breast tumors develop tamoxifen resistance and are eventually refractory to tamoxifen therapy. The molecular mechanisms underlying development of tamoxifen resistance have not been well established. Recently, we reported that breast cancer cells with high levels of ER-α36, a variant of ER-α, were resistant to tamoxifen and knockdown of ER-α36 expression in tamoxifen resistant cells with the shRNA method restored tamoxifen sensitivity, indicating that gained ER-α36 expression is one of the underlying mechanisms of tamoxifen resistance. Here, we found that tamoxifen induced expression of ER-α36-EGFR/HER2 positive regulatory loops and tamoxifen resistant MCF7 cells (MCF7/TAM) expressed enhanced levels of the loops. Disruption of the ER-α36-EGFR/HER2 positive regulatory loops with the dual tyrosine kinase inhibitor Lapatinib or ER-α36 down-regulator Broussoflavonol B in tamoxifen resistant MCF7 cells restored tamoxifen sensitivity. In addition, we also found both Lapatinib and Broussoflavonol B increased the growth inhibitory activity of tamoxifen in tumorsphere cells derived from MCF7/TAM cells. Our results thus demonstrated that elevated expression of the ER-α36-EGFR/HER2 loops is one of the mechanisms by which ER-positive breast cancer cells escape tamoxifen therapy. Our results thus provided a rational to develop novel therapeutic approaches for tamoxifen resistant patients by targeting the ER-α36-EGFR/HER2 loops.     

Simultaneous quantification of GABAergic 3α,5α/3α,5β neuroactive steroids in human and rat serum

The 3α,5α- and 3α,5β-reduced derivatives of progesterone, deoxycorticosterone, dehydroepiandrosterone and testosterone enhance GABAergic neurotransmission and produce inhibitory neurobehavioral and anti-inflammatory effects. Despite substantial information on the progesterone derivative (3α,5α)-3-hydroxypregnan-20-one (3α,5α-THP, allopregnanolone), the physiological significance of the other endogenous GABAergic neuroactive steroids has remained elusive. Here, we describe the validation of a method using gas chromatography–mass spectrometry to simultaneously identify serum levels of the eight 3α,5α- and 3α,5β-reduced derivatives of progesterone, deoxycorticosterone, dehydroepiandrosterone and testosterone. The method shows specificity, sensitivity and enhanced throughput compared to other methods already available for neuroactive steroid quantification. Administration of pregnenolone to rats and progesterone to women produced selective effects on the 3α,5α- and 3α,5β-reduced neuroactive steroids, indicating differential regulation of their biosynthetic pathways. Pregnenolone administration increased serum levels of 3α,5α-THP (+1488%, p < 0.001), (3α,5α)-3,21-dihydroxypregnan-20-one (3α,5α-THDOC, +205%, p < 0.01), (3α,5α)-3-hydroxyandrostan-17-one (3α,5α-A, +216%, p < 0.001), (3α,5α,17β)-androstane-3,17-diol (3α,5α-A-diol, +190%, p < 0.01). (3α,5β)-3-hydroxypregnan-20-one (3α,5β-THP) and (3α,5β)-3-hydroxyandrostan-17-one (3α,5β-A) were not altered, while (3α,5β)-3,21-dihydroxypregnan-20-one (3α,5β-THDOC) and (3α,5β,17β)-androstane-3,17-diol (3α,5β-A-diol) were increased from undetectable levels to 271 ± 100 and 2.4 ± 0.9 pg ± SEM, respectively (5/8 rats). Progesterone administration increased serum levels of 3α,5α-THP (+1806%, p < 0.0001), 3α,5β-THP (+575%, p < 0.001), 3α,5α-THDOC (+309%, p < 0.001). 3α,5β-THDOC levels were increased by 307%, although this increase was not significant because this steroid was detected only in 3/16 control subjects. Levels of 3α,5α-A, 3α,5β-A and pregnenolone were not altered. This method can be used to investigate the physiological and pathological role of neuroactive steroids and to develop biomarkers and new therapeutics for neurological and psychiatric disorders.     

The involvement of cytochrome b5 in 5β-cholestane-3α,7α,12α-triol 25-hydroxylation and taurodeoxycholate 7α-hydroxylation of rat liver

Role of cytochrome b₅ in NADPH-supported 5β-cholestane-3α,7α,12α-triol 25-hydroxylation and taurodeoxycholate 7α-hydroxylation of rat liver microsomes was investigated using highly purified antibodies against cytochrome b₅. Anti-b₅ antibody strongly inhibited both hydroxylation reactions indicating that cytochrome b₅ is a functional component in these steroid hydroxylation systems. It was shown that the involvement of cytochrome b₅ in these systems could be altered by the conditions of the reaction systems.     

Studies on the effect of 5α-androstane-3α, 17α-diol in the canine prostate in vivo

Adult beagle dogs, castrated one month previously, were treated with 5α-androstane-3α, 17α-diol (total dose 300 mg) over a period of one month. Examination of the prostate after treatment showed no significant change in size, weight or histological appearance from the castrate dog prostate. Subsequent administration of 5α-androstane-3α, 17β-diol (300 mg) over the same period of time resulted in restoration of the prostate size, weight and histological appearance to that of the normal intact dog prostate. It is concluded that exogenously administered 5α-androstane-3α, 17α-diol does not promote prostatic growth in the castrate beagle dog.     

The identification and characterization of the c19o3 steroid metabolites of 5α-androstane-3β, 17β-diol produced by the canine prostate: 5α-androstane-3β, 6α, 17β-triol and 5α-androstane-3β, 7α, 17β-triol

This study has identified the polar metabolites of 5α-androstane-3β, 17β-diol(3β-diol) produced by the canine prostate. The major metabolite is 5α-androstane-3β, 7α, 17β-triol (7α-triol) accounting for approximately 80% of the total polar metabolites of 3β-diol. The remaining 20% is accounted for exclusively by another triol, 5α-androstane-3β, 6α, 17β-triol(6α-triol). This study has also characterized two enzymatic hydroxylases responsible for respective triol formation: 5α-androstane-3β, 17β-diol 6α-hydroxylase (6α-hydroxylase) and 5α-androstane-3β, 17β-diol 7α-hydroxylase (7α-hydroxylase). Both of these irreversible hydroxylases are located in the particulate fraction of the prostate and can utilize either NADH or NADPH as cofactor. Several $\text{in vitro}$ steroid inhibitors of these hydroxylases were identified including cholesterol, estradiol and diethylstilbestrol. Neither of the hydroxylases were found to be decreased by castration (3 months) when expressed as activity/DNA. Using a variety of C₁₉ androstane substrates, 6α- and 7α-triol were found to be major components of the total 3β-hydroxy-5α-androstane metabolites produced by the canine prostate.     

Crystal structure of α/β-galactoside α2,3-sialyltransferase from a luminous marine bacterium, Photobacterium phosphoreum

α/β-Galactoside α2,3-sialyltransferase produced by Photobacterium phosphoreum JT-ISH-467 is a unique enzyme that catalyzes the transfer of N-acetylneuraminic acid residue from cytidine monophosphate N-acetylneuraminic acid to acceptor carbohydrate groups. The enzyme recognizes both mono- and di-saccharides as acceptor substrates, and can transfer Neu5Ac to both α-galactoside and β-galactoside, efficiently. To elucidate the structural basis for the broad acceptor substrate specificity, we determined the crystal structure of the α2,3-sialyltransferase in complex with CMP. The overall structure belongs to the glycosyltransferase-B structural group. We could model a reasonable active conformation structure based on the crystal structure. The predicted structure suggested that the broad substrate specificity could be attributed to the wider entrance of the acceptor substrate binding site.     

Improved synthesis of 3α,7α,12α,24ξ -tetrahydroxy-5 β -cholestan-26-oic acid

This paper describes three simple and short methods for the conversion of cholic acid into cholylaldehyde with protected hydroxyl groups. The first method involves lithium aluminum hydride reduction of the tetrahydropyranyl ether of methyl cholate and oxidation of the resulting primary alcohol with pyridinium chlorochromate. The second method employs diborane for the reduction of the -COOH group to the -CH₂OH group, while the third method involves the reduction of 3α, 7α, 12α -triformyloxy-5β -cholan-24-oic acid (as the acid chloride) directly into 3α, 7α, 12α -triformyloxy-5β -cholan-24-al with TMA-ferride (tetramethylammonium hydridoirontetracarbonyl). The aldehyde obtained by any of the above methods underwent smooth Reformatsky reaction with ethyl α -bromopropionate to yield 3α, 7α, 12α, 24ξ -tetrahydroxy-5β -cholestan-26-oic acid.