Girgert Rainer, Bartsch Christian, Hill Steven M, Kreienberg Rolf, Hanf Volker
Dept. of Gynecology and Obstetrics, University of Ulm, Prittwitzstrasse 43, D-89075 Ulm, Germany.
Neuro Endocrinol Lett. 2003 Dec;24(6):440-4.
Detection of the antiestrogenic effect of melatonin on various breast cancer cell lines and its dependence of the differential expression of estrogen receptors (ERalpha and ERbeta) and melatonin receptors (mt1 and RZRalpha).
Dose-response curves of estradiol were determined in 6 different breast cancer cell lines using a colorimetric proliferation assay in the absence or presence of various melatonin concentrations.
In order to detect the minor growth inhibitory effect of melatonin, a simple yet novel approach was employed: instead of incubating cells at single estradiol-concentrations at increasing melatonin levels, breast cancer cells were grown in microwell-plates for 4 days at increasing concentrations of estradiol (10(-12)M - 10(-10)M) in the absence or presence of melatonin (10(-9)M - 10(-8)M). Cell number was determined using Alamar blue and colorimetry. RT-PCR was performed for the expression of ERalpha, ERbeta, RZRalpha and mt1.
Melatonin at concentrations of 10(-9)M and 5 x 10(-9)M shifted the dose-response curves of estradiol to higher concentrations. Responsiveness to melatonin depended on expression of ERalpha but not on ERbeta. mRNA of ERbeta was not detectable in the breast cancer cell lines used. Only small amounts of mt1 transcripts were detectable in MCF-7 cells of one source. In MCF-7 cells transfected with the mt1 gene and in an ovarian cancer cell line mt1 was expressed at significant levels. RZRalpha was expressed in all tested cell lines at different amounts.
The growth of all ERalpha-positive breast cancer cell lines can be inhibited by melatonin. The effect in most cell lines is weak yet clearly reproducible. RZRalpha clearly contributes to the growth inhibitory effect of melatonin.
检测褪黑素对各种乳腺癌细胞系的抗雌激素作用及其对雌激素受体（ERα和ERβ）和褪黑素受体（mt1和RZRα）差异表达的依赖性。
在6种不同的乳腺癌细胞系中，通过比色增殖测定法，在存在或不存在不同浓度褪黑素的情况下，测定雌二醇的剂量反应曲线。
为检测褪黑素轻微的生长抑制作用，采用了一种简单而新颖的方法：不是在增加褪黑素水平时将细胞在单一雌二醇浓度下孵育，而是将乳腺癌细胞在微孔板中于不存在或存在褪黑素（10⁻⁹M - 10⁻⁸M）的情况下，在增加的雌二醇浓度（10⁻¹²M - 10⁻¹⁰M）下培养4天。使用阿拉玛蓝和比色法测定细胞数量。对ERα、ERβ、RZRα和mt1的表达进行逆转录聚合酶链反应（RT-PCR）。
浓度为10⁻⁹M和5×10⁻⁹M的褪黑素将雌二醇的剂量反应曲线向更高浓度偏移。对褪黑素的反应性取决于ERα的表达，而不取决于ERβ。在所使用的乳腺癌细胞系中未检测到ERβ的mRNA。在一个来源的MCF-7细胞中仅可检测到少量的mt1转录本。在转染了mt1基因的MCF-7细胞和一种卵巢癌细胞系中，mt1以显著水平表达。RZRα在所有测试细胞系中以不同量表达。
褪黑素可抑制所有ERα阳性乳腺癌细胞系的生长。在大多数细胞系中该作用较弱但明显可重复。RZRα明显有助于褪黑素的生长抑制作用。
