Angelucci Cristiana, Iacopino Fortunata, Lama Gina, Capucci Susanna, Zelano Giovanni, Boca Manila, Pistilli Alessandra, Sica Gigliola
Institute of Histology and Embryology, Faculty of Medicine, Catholic University of the Sacred Heart, Largo F. Vito 1, 00168 Rome, Itaty.
Anticancer Res. 2004 Sep-Oct;24(5A):2729-38.
In this study we confirmed the ability of a Gonadotropin Releasing Hormone (GnRH) agonist, leuprorelin acetate (LA), to counteract or even suppress the 5alpha-dihydrotestosterone (DHT)-stimulated growth of androgen-sensitive prostate cancer cells (LNCaP). Since the cellular mechanisms mediating this effect are not well defined, we investigated the activity of LA, also in combination with DHT or with cyproterone acetate (CA), on the expression of genes (bcl-2, bax, c-myc) which may contribute to the proliferative behaviour of LNCaP cells. In addition, experiments aimed to evaluate the action of the analogue on apoptosis were performed.
Gene expression was evaluated by RT-PCR and Western blotting on cells treated with LA (10(-11) or 10(-6) M), alone or combined with 10(-9) M DHT or 10(-7) M CA. The occurrence of apoptosis following treatment with LA (10(-11), 10(-6) or 10(-5) M), alone or combined with 10(-9) M DHT, was assessed by DNA fragmentation analysis.
Both the mRNA and protein of the anti-apoptotic gene bcl-2 were induced (30-125%) by DHT after 24-144 h. LA decreased bcl-2 mRNA (10-40%), while it did not unequivocally affect protein expression. The analogue always reduced (13-74%) both mRNA and protein levels obtained under DHT treatment. The mRNA and protein of the pro-apoptotic gene bax were down-regulated by DHT (15-40%), while LA generally induced bax protein but not its mRNA. LA counteracted DHT activity, even increasing bax protein levels over the controls. c-myc mRNA and protein were enhanced by DHT (15-45%) but down-regulated by LA (10-40%). Once more, the androgen effect was antagonized by LA, sometimes reducing c-myc content below the controls. CA produced the most similar effects to those triggered by DHT. The hormonal treatment did not induce any DNA fragmentation.
In spite of gene modulation, apoptosis was not observed under LA treatment, in agreement with the lack of a cell growth effect when the analogue was used alone. Nevertheless, the observed changes in gene expression may be directly or indirectly involved in the antiproliferative effect of LA on androgen-stimulated cells.
在本研究中，我们证实了促性腺激素释放激素（GnRH）激动剂醋酸亮丙瑞林（LA）能够抵消甚至抑制5α - 双氢睾酮（DHT）刺激的雄激素敏感性前列腺癌细胞（LNCaP）的生长。由于介导这种效应的细胞机制尚未明确，我们研究了LA单独或与DHT或醋酸环丙孕酮（CA）联合使用时，对可能影响LNCaP细胞增殖行为的基因（bcl - 2、bax、c - myc）表达的活性。此外，还进行了旨在评估该类似物对细胞凋亡作用的实验。
通过RT - PCR和蛋白质印迹法评估用LA（10⁻¹¹或10⁻⁶ M）单独或与10⁻⁹ M DHT或10⁻⁷ M CA联合处理的细胞中的基因表达。通过DNA片段化分析评估用LA（10⁻¹¹、10⁻⁶或10⁻⁵ M）单独或与10⁻⁹ M DHT联合处理后细胞凋亡的发生情况。
抗凋亡基因bcl - 2的mRNA和蛋白质在24 - 144小时后均被DHT诱导（30 - 125%）。LA降低了bcl - 2的mRNA（10 - 40%），而对蛋白质表达没有明确影响。该类似物总是降低（13 - 74%）在DHT处理下获得的mRNA和蛋白质水平。促凋亡基因bax的mRNA和蛋白质被DHT下调（15 - 40%），而LA通常诱导bax蛋白质但不诱导其mRNA。LA抵消了DHT的活性，甚至使bax蛋白质水平高于对照组。c - myc的mRNA和蛋白质被DHT增强（15 - 45%）但被LA下调（10 - 40%）。同样，LA拮抗了雄激素的作用，有时使c - myc含量低于对照组。CA产生的效应与DHT引发的效应最为相似。激素处理未诱导任何DNA片段化。
尽管存在基因调节，但在LA处理下未观察到细胞凋亡，这与单独使用该类似物时缺乏细胞生长效应一致。然而，观察到的基因表达变化可能直接或间接参与了LA对雄激素刺激细胞的抗增殖作用。
