丁酸钠抑制芳香酶肿瘤相关性启动子激活的分子机制

背景与目的：组蛋白去乙酰化酶（histone deacetylase,HDAC）抑制剂丁酸钠特异性阻断肿瘤条件液诱导的启动子PⅡ、PⅠ.3异常激活,本文旨在通过探讨丁酸钠作用的关键环节,来了解乳腺癌发病中PⅡ、PⅠ.3异常激活的分子机制。方法：从乳腺癌标本中获取原代脂肪成纤维细胞进行原代培养,加入人乳腺癌细胞MCF-7的条件培养液,作为芳香酶肿瘤相关性启动子（PⅡ、PⅠ.3）激活的研究细胞模型。用免疫印迹、电泳迁移率变动分析等方法观察丁酸钠对PⅡ、PⅠ.3活性的影响及其分子机制。结果：在原代脂肪成纤维细胞中,ATF-2蛋白是CREB/ATF转录因子家族主要的调节亚型,并组成性结合于CRE(-199/-211bp)位点,丁酸钠的关键作用环节在于抑制ATF-2蛋白的磷酸化,进而抑制C/EBPβ、CBP、磷酸化ATF-2蛋白转录复合物的形成。结论：ATF-2磷酸化是PⅡ、PⅠ.3激活的关键分子机制。     

Aromatase gene expression and its modifying factors in breast tumor tissue

Aromatase (CYP 19) gene expression was studied in 70 breast tumors. When RNA-dot-blot or rt-polymerase chain reaction were used expression frequency was 60.4 and 91.7%, respectively. An analysis of individual variants of non-coding exon of aromatase gene confirmed that, unlike normal mammary tissue, tumor switched from activation of exon I.4 ("sensitive" to glucocorticoids) to exons II ("sensitive" to cAMP) or I.3. This difference was relatively somewhat more pronounced in the Russian material. Direct correlation between aromatase enzymatic activity and expression of exons II and I.3 in tumor tissue appeared more significant than that of aromatase gene coding site. An evaluation of the expression of adenylate cyclase G-protein alpha-subunit genes established an inverse correlation between expression of Gi2a and exon I.3. Breast tumors with elevated basal aromatase activity were more sensitive to aromatase inhibitors (letrozole, 4-OHA) in vitro although no relationship between use of CYP19 (aromatase) 5' exon variant and in vitro inhibition of aromatase was detected. A correlation was observed between expression of aromatase gene and variants of its 5' exon, on the one hand, and age, tumor grade, steroid receptor presence and tumor lymphocytic infiltration, on the other. To summarize, local estrogen production in breast tumor tissue is regulated by a wide range of factors expression both aromatase gene influencing and its enzymatic activity, thus providing leverage on both.     

Inhibitory Effects of Calcitriol on the Growth of MCF-7 Breast Cancer Xenografts in Nude Mice: Selective Modulation of Aromatase Expression in vivo

Calcitriol (1,25-dihydroxyvitamin D(3)), the hormonally active metabolite of vitamin D, exerts many anticancer effects in breast cancer (BCa) cells. We have previously shown using cell culture models that calcitriol acts as a selective aromatase modulator (SAM) and inhibits estrogen synthesis and signaling in BCa cells. We have now examined calcitriol effects in vivo on aromatase expression, estrogen signaling, and tumor growth when used alone and in combination with aromatase inhibitors (AIs). In immunocompromised mice bearing MCF-7 xenografts, increasing doses of calcitriol exhibited significant tumor inhibitory effects (~50% to 70% decrease in tumor volume). At the suboptimal doses tested, anastrozole and letrozole also caused significant tumor shrinkage when used individually. Although the combinations of calcitriol and the AIs caused a statistically significant increase in tumor inhibition in comparison to the single agents, the cooperative interaction between these agents appeared to be minimal at the doses tested. Calcitriol decreased aromatase expression in the xenograft tumors. Importantly, calcitriol also acted as a SAM in the mouse, decreasing aromatase expression in the mammary adipose tissue, while increasing it in bone marrow cells and not altering it in the ovaries and uteri. As a result, calcitriol significantly reduced estrogen levels in the xenograft tumors and surrounding breast adipose tissue. In addition, calcitriol inhibited estrogen signaling by decreasing tumor ERα levels. Changes in tumor gene expression revealed the suppressive effects of calcitriol on inflammatory and growth signaling pathways and demonstrated cooperative interactions between calcitriol and AIs to modulate gene expression. We hypothesize that cumulatively these calcitriol actions would contribute to a beneficial effect when calcitriol is combined with an AI in the treatment of BCa.     

Characterisation of aromatase expression in the human adipocyte cell line SGBS

Aromatase is a member of the cytochrome P450 superfamily of enzymes which catalyses the rate-limiting step in the biosynthesis of estrogens. A number of clinical studies have highlighted the importance of local estrogen production in adipose tissue. In particular, in the postmenopausal woman, the degree of her estrogenization is mainly determined by the extent of her adiposity and it is this extragonadal source of estrogen that likely contributes to breast cancer development and progression. The mechanisms regulating aromatase expression in adipose tissue however, have not been fully elucidated. In this study, we have characterised the expression of aromatase and its activity in a human preadipocyte cell strain, SGBS. Aromatase is expressed in SGBS cells and its expression and activity are strongly stimulated by forskolin (FSK) and phorbol 12-myristate-13-acetate (PMA) treatment. Consistent with this, FSK and PMA treatment also increased activation of the proximal aromatase promoter, promoter II. These findings mimic those that have previously been shown in isolated primary human preadipocytes. These data suggest that SGBS cells are a valuable model with which to further elucidate the mechanisms regulating aromatase expression, and therefore local estrogen synthesis in human adipose tissue.     

Interactions between prostaglandin E(2), liver receptor homologue-1, and aromatase in breast cancer

Local synthesis of estrogens within breast adipose tissue by cytochrome P450 aromatase contributes to the growth of postmenopausal breast cancers. One of the major stimulators of aromatase expression in breast is prostaglandin E(2) (PGE(2)) derived from tumorous epithelium and/or infiltrating macrophages. Recently, the orphan nuclear receptor, liver receptor homologue-1 (LRH-1), has also been shown to regulate aromatase expression in breast adipose tissue. We therefore examined the expression of, and correlations between, aromatase and LRH-1 mRNA in a panel of breast carcinoma tissues and adjacent adipose tissue. LRH-1 mRNA expression was low in normal breast tissue but markedly elevated in both breast carcinoma tissue and adipose tissue surrounding the tumor invasion (thereby paralleling aromatase expression). Laser capture microdissection localized the site of LRH-1 expression to tumor epithelial cells but not to intratumoral stromal cells. A strong correlation between LRH-1 and aromatase mRNA levels was observed in tumor-containing adipose tissue but not in tumor tissue. Ectopic expression of LRH-1 in primary human adipose stromal cells strongly activated endogenous aromatase mRNA expression and enzyme activity. Finally, treatment of adipose stromal cells with PGE(2) induced expression of both LRH-1 and aromatase. We suggest that PGE(2) derived from breast tumor tissue may increase aromatase expression in the surrounding adipose stroma in part by inducing LRH-1 in these cells. The roles of LRH-1 in breast cancer proliferation merit further study.     

Regulation of aromatase promoter activity in human breast tissue by nuclear receptors

Using the yeast one-hybrid approach to screen a human breast tissue hybrid cDNA expression library, we have found that four orphan/nuclear receptors, ERRalpha-1, EAR-2, COUP-TFI (EAR-3), and RARgamma, bind to the silencer (S1) region of the human aromatase gene. S1 down regulates promoters I.3 and II of the human aromatase gene. In this study, the interaction of EAR-2, COUP-TFI, and RARgamma with S1 was confirmed by DNA mobility shift analysis. In contrast to the findings that ERRalpha-1 behaves as a positive regulatory factor, these three nuclear receptors were found, by mammalian cell transfection experiments, to act as negative regulatory factors by binding to S1. Furthermore, the negative action of these three nuclear receptors could override the positive effect of ERRalpha-1. RT-PCR analysis of 11 cell lines and 55 human breast tumor specimens has shown that these nuclear receptors are expressed in human breast tissue. Since EAR-2, COUP-TFI, and RARgamma are expressed at high levels, it is likely that S1 is a negative regulatory element that suppresses aromatase promoters I.3 and II in normal breast tissue. In cancer tissue, S1 may function as a positive element since ERRalpha-1 is expressed, but EAR-2 and RARgamma are only present in a small number of tumor specimens. This hypothesis is sustained by the finding that there is a weak inverse correlation between the expression of COUP-TFI and that of aromatase in breast tumor tissue. Our studies have revealed that estrogen receptor alpha (ERalpha) can also bind to S1, in a ligand-dependent manner. By binding to S1, ERalpha down-regulates the aromatase promoter activity. These results demonstrate that nuclear receptors play important roles in modulating aromatase expression in human breast tissue.     

Role of cyclic AMP responsive element in the UVB induction of cyclooxygenase-2 transcription in human keratinocytes

It has been shown that UVB irradiation induces expression of COX-2 and up-regulation of COX-2 plays a functional role in UVB tumor promotion. In this study, we examined the cis-elements in the human COX-2 promoter that may be responsible for the UVB induction of COX-2. Analyses with the COX-2 promoter region revealed that the cyclic AMP responsive element near the TATA box was essential for both basal and UVB induced COX-2 expression. This was further supported by studies using a dominant negative mutant of CREB, which strongly inhibited the activity of COX-2 promoter. Electrophoretic mobility shift assays indicated that CREB and ATF-1 were the major proteins binding to the COX-2 CRE. CREB and ATF-1 were phosphorylated upon UVB treatment, and SB202190, a p38 MAPK inhibitor, decreased the phosphorylation of CREB/ATF-1 and suppressed COX-2 promoter activity. In contrast, treatment with forskolin, an activator of adenylyl cyclase, led to phosphorylation of CREB and ATF-1 and activation of COX-2 promoter. Finally, enhanced binding of phospho-CREB/ATF-1 to the COX-2 CRE was observed after UVB induction. Thus, one signaling pathway for UVB induction of human COX-2 involves activation of p38, subsequent phosphorylation of CREB/ATF-1, and activation of the COX-2 CRE through enhanced binding of phosphorylated CREB/ATF-1.     

Regulation of Cox-2 by Cyclic AMP Response Element Binding Protein in Prostate Cancer: Potential Role for Nexrutine

We recently showed that Nexrutine^R, a Phellodendron amurense bark extract, suppresses proliferation of prostate cancer cell lines and tumor development in the transgenic adenocarcinoma of mouse prostate (TRAMP) model. Our data also indicate that the anti-proliferative effects of Nexrutine^R are emediated in part by Akt and Cyclic AMP response element binding protein (CREB).Cyclooxygenase (Cox-2), a pro-inflammatory mediator, is a CREB target that induces prostaglandin E₂ (PGE₂) and suppresses apoptosis. Treatment of LNCaP cells with Nexrutine^R reduced tumor necrosis factor α-induced enzymatic as well as promoter activities of Cox-2. Nexrutine^R also reduced the expression and promoter activity of Cox-2 in PC-3 cells that express high constitutive levels of Cox-2. Deletion analysis coupled with mutational analysis of the Cox-2 promoter identified CRE as being sufficient for mediating Nexrutine^R response. Immunohistochemical analysis of human prostate tumors show increased expression of CREB and DNA binding activity in high-grade tumors (three-fold higher in human prostate tumors compared to normal prostate; P = .01). We have identified CREB-mediated activation of Cox-2 as a potential signaling pathway in prostate cancer which can be blocked with a nontoxic, cost-effective dietary supplement like Nexrutine^R, demonstrating a prospective for development of Nexrutine^R for prostate cancer management.