Effect of antiprogestins and tamoxifen on growh inhibition of MCF-7 human breast cancer cells in nude mice

This is the first report demonstrating an in vivo antitumor activity of antiprogestins (mifepristone, onapristone) alone and in combination with tamoxifen in the MCF-7 human breast cancer model. The MCF-7 cells produced progressive growing tumors in female nude mice supplemented with 17-estradiol. Tumor regression was observed following either estrogen ablation alone or estrogen ablation in combination with tamoxifen. Monotherapy with tamoxifen or antiprogestins caused a retardation of estrogen-induced tumor progression. Complete inhibition or prevention of tumor growth occurred as a result of simultaneous administration of mifepristone and tamoxifen. The addition of mifepristone in this combination treatment was also effective in delaying or preventing tumor escape (relapse) from the antiestrogenic (antitumor) effect of tamoxifen. These results suggest a potential clinical benefit of adding an antiprogestin to antiestrogen therapy of breast cancer patients.     

Association between intratumoral free and total VEGF, soluble VEGFR-1, VEGFR-2 and prognosis in breast cancer

Vascular endothelial growth factor (VEGF) receptors consist of three cell-membrane type receptors (VEGFR-1, VEGFR-2 and VEGFR-3), and soluble form of VEGFR-1 (sVEGFR-1), an intrinsic negative counterpart of the VEGF. In this study, we measured intratumoral protein levels of free and total VEGF, VEGFR-2 and sVEGFR-1 from 202 primary breast cancer tissues and examined their prognostic values. A significant inverse correlation was found between free or total VEGF and oestrogen receptor (ER) status (P=0.042 and 0.032, respectively). A univariate analysis showed that low sVEGFR-1 and high total VEGF were significantly associated with poor prognosis in disease-free survival (DFS) and overall survival (OS). The ratio of sVEGFR-1 to total VEGF was a strong prognostic indicator (DFS: P=0.008; OS: P=0.0002). A multivariate analysis confirmed the independent prognostic values of total VEGF and the ratio of sVEGFR-1 to total VEGF. In subgroup analysis, total VEGF was a significant prognostic indicator for ER-positive tumours but not for ER-negative tumours, whereas sVEGFR-1 was significant for ER-negative tumours but not for ER-positive tumours. In conclusion, the intratumoral sVEGFR-1 level, VEGF level and the ratio of sVEGFR-1 to total VEGF are potent prognostic indicators of primary breast cancer, and might be relevant to ER status.     

Biological differences among MCF-7 human breast cancer cell lines from different laboratories

Summary MCF-7 human breast cancer cells are used widely for studies of tumor biology and hormone mechanism of action. Conflicting results have often been obtained in studies reported from different laboratories. In this report several biological properties were studied in four MCF-7 cell lines obtained from different laboratories. MCF-7 (ATCC), MCF-7, MCF-7 (KO), and MCF-7 (S) demonstrated similar morphology in monolayer culture. Chromosome analysis revealed that three of the lines shared several structural chromosome alterations and marker chromsomes; however, MCF-7 (ATCC) was distinctly different with virtually no chromosomal alterations shared in common with the other lines. All four lines contained variable amounts of estrogen receptor (ER) and progesterone receptor (PgR). The growth rate of MCF-7 (ATCC) was 50% slower than that of the other lines, and, unlike the other three lines, cell proliferation was unaffected by estrogen or antiestrogen treatment despite the presence of receptors. Cloning efficiency of the four lines varied over a 10-fold range. Tumorigenicity in athymic nude mice also varied considerably among these lines. MCF-7 (ATCC) grew well in ovariectomized nude mice, while the other lines required estrogen supplementation. MCF-7 (S) and MCF-7 grew rapidly with estrogen supplementation; MCF-7 (KO) grew very slowly. Antiestrogen therapy inhibited growth of MCF-7, MCF-7 (S), and MCF-7 (KO) tumors, but it had no effect on MCF-7 (ATCC). These data demonstrate that MCF-7 lines from different laboratories may have unique biological properties, despite having a similar karyotype (MCF-7, MCF-7 (S), MCF-7 (KO)). The fundamental differences in karyotype and biological properties of the MCF-7 (ATCC), and the previously reported differences in DNA restriction fragment polymorphism analyses, demonstrate that this line is derived from an entirely different patient. Investigators should carefully document the source and identity of MCF-7 cells used in published experiments.     

人肝细胞癌VEGF及受体的表达与临床病理的关系

目的:分析VEGF及其受体flt-1/flk-1蛋白表达与肝细胞癌临床病理的关系.方法:应用ABC免疫组化染色法,检测60例肝细胞癌中VEGF、flk-1、flt-1蛋白表达情况,并分析它们与肝细胞癌临床病理的关系.结果:60例肝细胞癌组织中VEGF、flk-1、flt-1蛋白的阳性表达率分别为81.3%、88.3%、80.0%,均明显高于正常肝组织(P＜0.05);并且VEGF蛋白表达与肿瘤的病理分级、有无包膜、瘤栓、直径大小均有相关性.结论:VEGF蛋白表达水平越高,细胞恶性程度及侵袭、转移能力越强,并且VEGF与flt-1/flk-1在肿瘤形成过程中具有协同作用.     

Long-term adjuvant tamoxifen therapy for breast cancer

Tamoxifen (ICI46,474) is a competitive inhibitor of estrogen action which has found ubiquitous application in the treatment of breast cancer. The drug is the front line endocrine therapy for breast cancer and is the proven treatment of choice for the adjuvant therapy of postmenopausal women with node-positive disease. Tamoxifen is available for the treatment of premenopausal patients with advanced disease, and is being evaluated in clinical trials as an adjuvant therapy for premenopausal patients with either node-positive or node-negative disease. Laboratory studies demonstrate that tamoxifen is a tumoristatic agent and long-term treatment strategies (chemosuppression) should be considered to apply the antiestrogen to its maximal therapeutic advantage. Optimal therapy with tamoxifen may also be achieved by treatment strategies to lower circulating estrogen levels in the premenopausal patient.Tamoxifen is a well tolerated drug, and long-term therapy does not appear to induce metabolic tolerance. Concerns about premature osteoporosis or cardiovascular disease appear to be unfounded because tamoxifen has an appropriate level of target site-directed estrogenic activity. Isolated reports about the growth or appearance of endometrial carcinoma during long-term adjuvant tamoxifen therapy must be balanced against the risks of withholding treatment to patients with a fatal disease.     

重组人IL-2抑制乳腺癌细胞增殖及其机制的研究

目的：观察重组人IL-2对体外培养乳腺癌细胞生长的影响,及对洛铂体外抑瘤效果的影响。方法：选择乳腺癌细胞株MCF-7和MDA-MB-231作为实验对象。利用MTT实验检测重组人IL-2和对照药物洛铂对肿瘤细胞生长的抑制率,利用RT-PCR方法分析MCF-7细胞中Syk基因在不同处理因素作用下表达情况。结果：洛铂能明显抑制两种乳腺癌细胞体外生长,重组人IL-2对两种癌细胞有一定的抑制作用。重组人IL-2和洛铂同时处理时,对肿瘤细胞的抑制率比单独应用洛铂时明显升高,并且同时处理时MCF-7细胞中Syk mRNA表达水平比洛泊单独处理时高。结论：重组人IL-2能增强洛铂对乳腺癌细胞生长抑制效果。重组人IL-2单独作用乳腺癌细胞时也有一定的抑制效果,针对MCF-7细胞,重组人IL-2刺激信号传递可能有Syk参与。提示在体内时IL-2不仅对机体免疫细胞起重要的刺激激活作用,还可能对瘤细胞本身也有抑制作用。     

Significance of vascular endothelial growth factor (VEGF)/soluble VEGF receptor-1 relationship in breast cancer

Angiogenesis, the formation of new blood vessels, is controlled by a balance between positive and negative endothelial regulatory factors. Soluble vascular endothelial growth factor receptor-1 (sVEGFR1), a naturally occurring soluble form of VEGFR1, is a negative counterpart of the vascular endothelial growth factor (VEGF) signaling pathway, which has been characterized as one of the most important endothelial regulators in human tumor angiogenesis. In our study, we examined the expression of sVEGFR1 in 110 primary breast carcinomas, and assessed its clinical significance. Ninety-four of 110 tumors showed > or = 0.1 ng/mg protein of sVEGFR1 (range:0. 1-6.9 ng/mg protein; median: 1.03 ng/mg protein) as determined by a specific enzyme-linked immunosorbent assay (ELISA). Immunoblot analysis confirmed the presence of sVEGFR1 in breast tumor tissues. The levels of sVEGFR1 were correlated significantly with the levels of VEGF. There was no significant correlation between the levels of sVEGFR1 and any clinico-pathological factors including age, menopause, nodal involvement and hormone receptor status. A univariate prognosis analysis showed that the intratumoral VEGF status, as determined by ELISA, was a significant prognostic indicator, but sVEGFR1 status was not. In the combined analysis, however, the ratio of sVEGFR1 and VEGF levels provided more statistically significant prognostic value than VEGF status alone. Tumors in which the sVEGFR1 levels exceeded VEGF levels 10-fold had a markedly favorable prognosis. Multivariate analysis also demonstrated that the ratio of sVEGFR1 and VEGF was an independent prognostic indicator after nodal status. In conclusion, sVEGFR1, an intrinsic inhibitor of VEGF, frequently co-expressed with VEGF in primary breast cancer tissues. The intratumoral balance between sVEGFR1 and VEGF levels might be crucial for the progression of breast cancer.     

VEGF、VEGF-C、flt-4在乳腺癌组织中的表达及临床病理分析

目的：探讨乳腺癌组织中VEGF、VEGF-C、fit-4蛋白的表达水平、相互关系及临床意义，方法：采用免疫组织化学ABC法，检测82例乳腺癌石蜡标本中VEGF、VEGF-C、fit-4蛋白的表达水平。结果：乳腺癌标本中VEGF、VEGF-C、fit-4蛋白的阳性表达率分别为57．3％、46．3％、50．0％，VEGF-C与fit-4蛋白的表达呈正相关。但与乳腺癌组织病理学分型、肿瘤大小和分级无关。结论：VEGF-C、fit-4蛋白的表达与乳腺癌淋巴结转移有关，并可能协同作用于淋巴结转移过程。     

Preliminary research on dendritic cells loaded with resistant breast cancer antigens in breast cancer-bearing nude mice

Objective The aim of the study was to investigate the inhibitory effects of dendritic cells(DCs) loaded with resistant breast cancer antigens on breast cancer in nude mice. Methods A single-cell suspension was prepared from a primary breast cancer and chemotherapeutic drugs were screened using the ATP-PCA susceptibility testing system. Cancer cells were treated with 1/10 × IC50, 1/5 × IC50, 1/2 × IC50, 1 × IC50, and 2 × IC50 medium until their growth became steady in the 2 × IC50 medium. Peripheral blood mononuclear cells(PBMCs) were obtained from the peripheral blood of patients with leukapheresis. The obtained adherent cells were induced by granulocyte-macrophage colony-stimulating factor(GM-CSF) and interleukin-4(IL-4) to generate DCs, which carried resistant strain cell lysis compounds or non-treated cancer cell lysis compounds. The former mature DCs carried resistant breast tumor antigens. A breast tumor-bearing nude mouse model was established with these resistant strains and the mice were randomly divided in three groups. The mice in the treatment group were injected with DCs loaded with resistant breast cancer antigens. The control group consisted of mice injected with DCs loaded with primary tumor cell antigens and the blank group consisted of mice injected with the same volume of normal saline. Changes in the cancers were observed. Results After treatment with the effector cells, the cancer volume and weight were significantly different to those before treatment in every group of mice(P < 0.05). The tumor volume in the blank group was the largest(3.362 ± 0.068 cm3) and the tumor weight was 637.50 ± 59.398 mg. Compared to the blank group, the tumor volume in the experimental group was the smallest(1.273 ± 0.071 cm3) and the tumor weight was 206.81 ± 32.711 mg. Conclusion DCs loaded with resistant breast cancer antigens demonstrated a significant inhibition effect on the cancers of breast tumor-bearing nude mice.     

乳岩宁方联合依西美坦诱导绝经后裸鼠乳腺癌移植瘤细胞凋亡的实验研究

目的：探讨乳岩宁方药与依西美坦联合应用对乳腺癌的抑制作用及其作用机理.方法：采用双侧卵巢切除的方法制备绝经后裸鼠模型,将对数生长期的MCF-7细胞调整浓度为5×107/ml,每只接种于卵巢切除后的裸鼠右侧胸壁第二乳垫部皮下,0.2ml/只,待裸鼠长出肿瘤0.8 cm3后,将取得的瘤块切成1mm3大小,无菌条件下接种于裸小鼠右腋窝脂肪垫下,建立绝经后裸鼠移植瘤模型.选取荷瘤成功的裸鼠随机分为4组：对照组、依西美坦组、乳岩宁组、联合组（乳岩宁联合依西美坦）.记录各组裸鼠饮食、活动、皮毛色泽、死亡及体重变化情况;第21天,处死裸鼠取肿瘤,称重,计算抑瘤率;TUNEL法测肿瘤细胞凋亡.结果：体重：实验结束时,各用药组体重比较无显著差别,但是对比体重下降,联合组下降最少,为0.93±0.06g,依西美坦组下降最多,为1.61±0.12g,乳岩宁组为1.11±0.16g,各组间比较有统计学差异（P＜0.05）;瘤重：依西美坦组为0.197±0.021g、乳岩宁组为0.223±0.028g、联合组0.161±0.021g,肿瘤重量均低于对照组的0.298±0.022g,与对照组比较有非常显著差异（P＜0.01）;依西美坦组、乳岩宁组、联合组的抑瘤率分别为33.9％、25.2％、46.0％;TUNEL法检测裸鼠肿瘤组织细胞凋亡结果：凋亡指数（AI）：联合组最高为（39.37±3.12）％,依西美坦组（31.41±2.89）％,乳岩宁组（27.89±2.94）％,与对照组相比有统计学差异（P＜0.01）;联合组与单独用药组比较差异均有统计学意义（P＜0.01）.结论：乳岩宁对裸鼠MCF-7乳腺癌移植瘤有抑制作用,与依西美坦有协同作用,增加抑瘤率,其作用机制可能通过诱导肿瘤细胞凋亡来实现的.     

Inhibition of angiogenesis and tumour growth by VEGF121-toxin conjugate: differential effect on proliferating endothelial cells

Vascular endothelial growth factor (VEGF) plays an important role in tumour angiogenesis. VEGF binds to tyrosine kinase receptors, which are expressed almost exclusively on tumour endothelium. Therefore, VEGF can be used to target toxin molecules to tumour vessels for anti-angiogenic therapy. However, recent evidence suggests that VEGF can also bind in an isoform-specific fashion to a newly identified neuropilin-1 (NP-1) receptor. NP-1 is widely expressed in normal tissue and presents a potential target for unwanted toxicity. As a consequence, we investigated whether the VEGF121 isoform, which lacks the NP-1 binding domain, could be used to target toxin polypeptides to tumour vasculature. Treatment of endothelial cells with a VEGF121-diphtheria toxin (DT385) conjugate selectively inhibited proliferating endothelial cells, whereas confluent cultures were completely resistant to the construct. In addition, VEGF121-DT385 conjugate treatment completely prevented tumour cell induced angiogenesis in vivo. Most importantly, the conjugate inhibited tumour growth in athymic mice and induced tumour-specific vascular damage. There was also no apparent toxicity associated with the treatment. Our results suggest that proliferating endothelial cells are highly sensitive to VEGF121-toxin conjugates and that the binding to NP-1 receptors is not necessary for efficient inhibition of tumour growth.     

靶向VEGF的不对称siRNA抑制乳腺癌MCF-7细胞的增殖

目的:探讨靶向血管内皮生长因子(vascular endothelial growth factor,VEGF)基因的siRNA对乳腺癌MCF-7细胞的抑制效果。方法:设计靶向VEGF的4种小干扰RNA(small interfering RNA,siRNA),包括对称siRNA(siRNA21/21,siRNA23/23)与不对称siRNA(asymmetric siRNA,aiRNA;aiRNA21/23,aiRNA19/21)。siRNA转染入MCF-7细胞后,MTT及流式细胞术检测MCF-7细胞增殖及凋亡情况,RT-PCR、ELISA法检测MCF-7细胞中VEGF基因和蛋白的表达。结果:与对照组相比,aiRNA21/23、siRNA23/23、aiRNA19/21、siRNA21/21这4种siRNA都能有效抑制VEGF mRNA的表达[(71.4±5.01)%、(40.0±3.11)%、(37.2±2.79)%、(11.1±0.99)%vs(2.4±0.11)%,P<0.01],且抑制MCF-7细胞的增殖[(44.7±5.38)%、(38.5±5.67)%、(33.6±2.18)%、(33.1±3.18)%vs(2.2±0.28)%,P<0.01],其中以不对称aiRNA21/23的抑制效果最明显。与对照组比较,aiRNA21/23显著促进MCF-7细胞的凋亡[(49.9±4.02)%vs(4.7±0.91)%,P<0.01]。结论:靶向VEGF基因的siRNA可抑制MCF-7细胞的增殖、促进细胞凋亡,尤以不对称siRNA的效果最明显。     

Combined effects of 1,25-dihydroxyvitamin D3 and tamoxifen on the growth of MCF-7 and ZR-75-1 human breast cancer cells

Summary In the present study we assessed the effect of combined treatment with 1,25-dihydroxyvitamin D₃ (1,25-(OH)₂D₃) and tamoxifen (TAM) on the growth of estrogen-responsive (MCF-7) and estrogen-dependent (ZR-75-1) human breast cancer cells. Both basal and 17-estradiol (17-E₂)-stimulated growth were studied. 1,25-(OH)₂D₃ (10^–10 – 10^–7 M) time- and dose-dependently inhibited basal growth of MCF-7 cells, with growth arrest at 10^–7 M. Also, 17-E₂-stimulated growth of MCF-7 and ZR-75-1 cells was inhibited by 1,25-(OH)₂D₃ in a time- and dose-dependent manner. TAM inhibited 17-E₂-stimulated growth of both cell lines and at high concentration (10^–6 M) it also inhibited basal growth of MCF-7 cells. 10^–6 M TAM together with 1,25-(OH)₂D₃ resulted in a further inhibition of basal (MCF-7 cells) as well as 17-E₂-stimulated proliferation (MCF-7 and ZR-75-1 cells) compared to the inhibition by these agents alone. TAM in combination with 10^–7 M 1,25-(OH)₂D₃ resulted in growth arrest of 17-E₂-stimulated growth of MCF-7 cells. The inhibition of basal and 17-E₂-stimulated growth of MCF-7 cells was additive at early time points (4 days), but less than additive at later time points (8–10 days). It was demonstrated that with co-treatment of MCF-7 cells an equipotent inhibition of basal growth could be reached with lower concentrations of 1,25-(OH)₂D₃, compared to treatment with 1,25-(OH)₂D₃ alone. Studies with low concentrations (< 10^–7 M) of TAM revealed a partial estrogenic effect, i.e. stimulation of MCF-7 proliferation in the absence of 17-E₂. This effect, which may resemble TAM-induced tumor flare, was completely prevented by co-treatment with a low concentration of 1,25-(OH)₂D₃ (10^–9 M). Together, these results demonstrate the potent inhibition of breast cancer cell proliferation by 1,25-(OH)₂D₃ combined with TAM and indicate a potential benefit of combining these agents for the treatment of breast cancer.     

三苯氧胺诱导人乳腺癌MCF-7细胞株耐药及自噬的关系研究

背景与目的:三苯氧胺(tamoxifen)作为第一代选择性雌激素受体调节剂(selective estrogen receptor modulator,SERM)被广泛地应用于激素敏感型乳腺癌的内分泌一线治疗。三苯氧胺耐药的发生严重限制了临床治疗,是乳腺癌患者用药面临的重大难题,明确其耐药机制对乳腺癌的治疗有重要临床意义。本研究通过体外诱导人乳腺癌细胞MCF-7三苯氧胺耐药,探讨细胞产生三苯氧胺耐药时自噬水平的变化与MAPK家族蛋白细胞外信号调节激酶(extracellular signal-regulated kinase,ERK)蛋白表达量及磷酸化水平的变化。方法:浓度递增筛选法诱导MCF-7细胞耐药,透射电镜观察MCF-7细胞与耐药细胞内的自噬泡数量,CCK8法检测细胞增殖状态,应用Western blot检测LC3Ⅱ、ERK1/2、Phospho-ERK1/2蛋白的表达情况。结果:诱导的三苯氧胺耐药细胞株TR5达到5μmol/L的耐药浓度。TR5细胞内的自噬泡数量与LC3Ⅱ表达量明显高于MCF-7细胞。ERK蛋白在两种细胞中的表达量差异无统计学意义,但其在TR5中的磷酸化水平比MCF-7细胞高。结论:...     

miR -21通过调控 PTEN 介导乳腺癌他莫昔芬耐药的作用机制

目的：研究 miR -21在乳腺癌他莫昔芬耐药中的作用,寻求逆转他莫昔芬耐药的靶点。方法：荧光实时定量 PCR（RT - QPCR）检测乳腺癌 MCF -7细胞与乳腺癌他莫昔芬耐药细胞株 MCF -7/ TAM 细胞中miR -21的表达;将 miR -21 mimics 转染到 MCF -7细胞内,将 miR -21 inhibitors 转染到 MCF -7/ TAM 细胞内,并应用 RT - QPCR 分别检测细胞内 miR -21的表达变化,给予细胞4-羟他莫昔芬（4- hydroxytamoxifen, OHT）处理后,利用 MTT 法检测细胞增殖的变化;转染 MCF -7/ TAM 细胞 miR -21 inhibitors 及 mimics,利用RT - QPCR 检测细胞中 PTEN 的表达变化。结果：miR -21在 MCF -7/ TAM 细胞表达高于 MCF -7细胞,转染 miR -21 mimics 的 MCF -7细胞 miR -21高表达,PTEN 表达降低,转染 miR -21 inhibitors 的 MCF -7/TAM 细胞 miR -21表达降低,PTEN 表达增高;过表达 miR -21的 MCF -7细胞给予 OHT 处理后,细胞的增殖抑制不明显,而沉默 miR -21的 MCF -7/ TAM 细胞给予 OHT 处理后,细胞增殖明显受到阻滞;转染耐药细胞 miR -21 inhibitors 后,PTEN 表达增高,转染 miR -21 mimics 后,PTEN 表达降低。结论：miR -21通过调控PTEN 的表达在乳腺癌 TAM 耐药中发挥重要作用。     

Melatonin enhances the inhibitory effect of aminoglutethimide on aromatase activity in MCF-7 human breast cancer cells

Summary The inhibition of the aromatase-induced intratumoral estrogen synthesis is one of the main anticancer pharmacological strategies. The aim of this paper was to study if a melatonin pretreatment prior to aminoglutethimide increases the efficiency of the aromatase inhibitor used in treating breast cancer. Aminoglutethimide (100 μM) and melatonin (1 nM) significantly decreased cellular aromatase activity in unpretreated MCF−7 cells. A sequential regimen of melatonin (1 nM) followed 24 h later by aminoglutethimide (100 μM) induced a significantly higher decrease in MCF-7 cell aromatase activity to below the values obtained in unpretreated cells. Melatonin treatment inhibited aromatase mRNA expression in unpretreated cells and a sequential treatment of cells with melatonin followed by aminoglutethimide induced a significant inhibition in the aromatase mRNA expression as compared to cells exposed to the same doses of aminoglutethimide, but without melatonin pretreatment.  The present study demonstrates that a treatment with melatonin followed by aminoglutethimide is the most effective way of reducing the aromatase activity in the MCF-7 cell line. The aminoglutethimide inhibitory effect is more potent when MCF-7 cells are pre-exposed to melatonin. Our results suggest that melatonin pretreatment increases the reduction of the aromatase activity of cells exposed to aminoglutethimide as a result of the decrease in the aromatase mRNA expression. The findings presented here point to melatonin pretreatment as a novel and interesting means to increase the efficacy of competitive aromatase inhibitors used in treating breast cancer.     

Functional analysis of MKP-1 and MKP-2 in breast cancer tamoxifen sensitivity

Increased activation of ERK signaling has been reported in breast cancer models of acquired tamoxifen resistance. Here, we examined the expression of Mitogen-Activated Protein Kinase Phosphatases (MKPs) 1 and 2 following tamoxifen treatment and the effects of MKP-1/MKP-2 overexpression on tamoxifen sensitivity. Treatment of MCF7 breast cancer cells with tamoxifen increased MKP-2, but not MKP-1, protein levels. Overexpression of MKP-1 or MKP-2 inhibited estrogen-induced MCF7 cell proliferation compared to vector controls. MCF7-MKP-2 cells displayed significantly increased sensitivity to tamoxifen as compared to vector control or MCF7-MKP-1 cells. MKP-1 or MKP-2 overexpression eliminated ERK1/2 phosphorylation, suggesting that decreases in estrogen-induced proliferation of MKP-1 and MKP-2 overexpressing cells are due to ERK1/2 dephosphorylation. JNK1/2 activation was not detectable in any of these cells. These data suggest that tamoxifen-induced death of these cells is not dependent upon JNK signaling, but rather that ERK is the major MAPK driving their proliferation. MCF7-TAMR cells express higher levels of MKP-2 mRNA and protein than MCF7 cells. MKP-2 and phospho-ERK1/2 proteins are constitutively expressed in MCF7-TAMR cells, and activated JNK1/2 is not detectable. These data suggest that MKP-2 rather than MKP-1 is tamoxifen-regulated and that the elevated expression of MKP-2 in MCF7-TAMR cells potentially functions to restore tamoxifen sensitivity.     

Vascular endothelial growth factor (VEGF) levels and mutation of the BRCA1 gene in breast cancer patients

The aim of the study was to compare VEGF serum levels in breast cancer patients with and without BRCA1 gene mutation. We enrolled 80 patients, 22 premenopausal and 58 postmenopausal. We found statistically significant lower levels of VEGF in patients with BRCA1 gene mutation as compared with breast cancer patients without this mutation.     

Prolactin overexpression by MDA-MB-435 human breast cancer cells accelerates tumor growth

Prolactin (PRL) is an important hormone in mammary tumorigenesis in rodents but its involvement in human breast cancer has been controversial. A role for locally produced PRL in breast carcinogenesis is suggested by its mitogenic action on breast cancer cells and the expression of both PRL and its receptor (PRL-R) in breast carcinomas. Our objective was to examine whether PRL, overexpressed by breast cancer cells, forms an autocrine/paracrine loop that confers a growth advantage for tumors. MDA-MB-435 breast cancer cells overexpressing 23K human PRL were generated, and PRL production and secretion by the clones were confirmed by RT-PCR, western blotting, and the Nb2 bioassay; control clones contain vector only. In vitro the 23K PRL clones proliferated faster and expressed higher levels of the PRL-R protein than controls only when incubated in charcoal-stripped serum (CSS) devoid of lactogenic hormones. When injected into the mammary fatpad of female nude mice or subcutaneously into males, the PRL-overexpressing clones formed tumors that grew 2–4-fold faster than tumors derived from control clones or wild type MDA-MB-435 cells. Western analysis demonstrated significantly higher PRL, PRL-R, and bcl-2 levels in the tumors overexpressing PRL compared to control tumors. These data support a role for breast PRL as a growth/anti-apoptotic factor and suggest that it may serve as a novel therapeutic target for the treatment of breast cancer.     

Melatonin inhibits aromatase promoter expression by regulating cyclooxygenases expression and activity in breast cancer cells

Background:

Melatonin reduces the development of breast cancer interfering with oestrogen-signalling pathways, and also inhibits aromatase activity and expression. Our objective was to study the promoters through which melatonin modifies aromatase expression, evaluate the ability of melatonin to regulate cyclooxygenases and assess whether the effects of melatonin are related to its effects on intracellular cAMP, in MCF-7 cells.

Methods:

Total aromatase mRNA, aromatase mRNA promoter regions and cyclooxygenases mRNA expression were determined by real-time RT–PCR. PGE₂ and cAMP were measured by kits.

Results:

Melatonin downregulated the gene expression of the two major specific aromatase promoter regions, pII and pI.3, and also that of the aromatase promoter region pI.4. Melatonin 1 nM was able to counteract the stimulatory effect of tetradecanoyl phorbol acetate on PGE₂ production and inhibit COX-2 and COX-1 mRNA expression. Melatonin 1 nM elicited a parallel time-dependent decrease in both cyclic AMP formation and aromatase mRNA expression.

Conclusions:

This study shows that melatonin inhibits aromatase activity and expression by regulating the gene expression of specific aromatase promoter regions. A possible mechanism for these effects would be the regulation by melatonin of intracellular cAMP levels, mediated by an inhibition of cyclooxygenase activity and expression.