Jinno Hiromitsu, Steiner Melissa G, Nason-Burchenal Kathryn, Osborne Michael P, Telang Nitin T
Department of Surgery, Keio University School of Medicine, Tokyo 160-8582, Japan.
Int J Oncol. 2002 Jul;21(1):127-34.
Aberrant proliferation is an early-occurring event in vitro prior to tumorigenesis in vivo in the multistep process of carcinogenesis. Inhibition of aberrant proliferation therefore may represent a useful biomarker to evaluate the efficacy of chemopreventive agents. Retinoids have exhibited preventive efficacy in vitro and in vivo predominantly through the retinoic acid receptors (RARs) and the retinoid X receptors (RXRs). Clinically relevant biochemical and cellular mechanistic endpoints for chemopreventive effects of retinoids should provide novel biomarkers. The present study was designed to examine the preventive efficacy of natural retinoids, all-trans-retinoic acid (ATRA) and 9-cis-retinoic acid (9cisRA), and to identify the possible mechanisms for their effects using the HER-2/neu oncogene expressing preneoplastic human mammary epithelial 184-B5/HER cells. Seven-day treatment with ATRA and 9cisRA exhibited a dose-dependent growth inhibition. Long-term (21 days) treatment with IC20 doses of 50 nM ATRA and 100 nM 9cisRA inhibited anchorage-dependent colony forming efficiency by about 75.4% (p<0.01) and 84.9% (p<0.01), respectively. Cell cycle analysis revealed that a 24-h treatment with IC90 doses of 2 microM ATRA and 3 microM 9cisRA accumulates cells in the G0/G1 phase and inhibit S and/or G2/M phase of the cell cycle. ATRA and 9cisRA induced an 11-fold (p=0.03) and a 9-fold (p=0.04) increase in subG0/G1 (apoptotic) population relative to the solvent control, respectively. ATRA and 9cisRA induced 77% (p=0.01) and 51% (p=0.02) decrease in tyrosine kinase immunoreactivity, respectively. Similarly, the two retinoids caused almost a 50% (p=0.01) down-regulation of Bcl-2 immunoreactivity. Western blot analysis revealed that ATRA induced an increase in RARbeta expression and a decrease in RARgamma expression, while 9cisRA down-regulated RXRalpha expression. These data demonstrate that ATRA and 9cisRA may inhibit HER-2/neu induced aberrant proliferation in part by retarding cell cycle progression, down-regulating HER-2/neu-mediated signal transduction and inducing Bcl-2-dependent apoptosis through a retinoid receptor-mediated mechanism.
在癌症发生的多步骤过程中，异常增殖是体内肿瘤发生之前在体外就出现的早期事件。因此，抑制异常增殖可能是评估化学预防剂疗效的有用生物标志物。类视黄醇在体外和体内均已显示出预防功效，主要是通过维甲酸受体（RARs）和类视黄醇X受体（RXRs）。类视黄醇化学预防作用的临床相关生化和细胞机制终点应能提供新的生物标志物。本研究旨在检测天然类视黄醇全反式维甲酸（ATRA）和9-顺式维甲酸（9cisRA）的预防功效，并使用表达HER-2/neu癌基因的癌前人类乳腺上皮184-B5/HER细胞来确定其作用的可能机制。用ATRA和9cisRA进行为期7天的处理显示出剂量依赖性生长抑制。用50 nM ATRA和100 nM 9cisRA的IC20剂量进行长期（21天）处理，分别使贴壁依赖性集落形成效率抑制约75.4%（p<0.01）和84.9%（p<0.01）。细胞周期分析显示，用2 microM ATRA和3 microM 9cisRA的IC90剂量进行24小时处理会使细胞在G0/G1期积累，并抑制细胞周期的S期和/或G2/M期。相对于溶剂对照，ATRA和9cisRA分别使亚G0/G1（凋亡）细胞群增加了11倍（p=0.03）和9倍（p=0.04）。ATRA和9cisRA分别使酪氨酸激酶免疫反应性降低了77%（p=0.01）和51%（p=0.02）。同样，这两种类视黄醇使Bcl-2免疫反应性下调了近50%（p=0.01）。蛋白质印迹分析显示，ATRA诱导RARβ表达增加和RARγ表达减少，而9cisRA下调RXRα表达。这些数据表明，ATRA和9cisRA可能部分通过延缓细胞周期进程、下调HER-2/neu介导的信号转导以及通过类视黄醇受体介导的机制诱导Bcl-2依赖性凋亡来抑制HER-2/neu诱导的异常增殖。
