Apoptosis-related gene expression affected by a GnRH analogue without induction of programmed cell death in LNCaP cells.
BACKGROUND
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.


METHODS
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.


RESULTS
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.


CONCLUSIONS
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.
Angelucci
Cristiana
C
Institute of Histology and Embryology, Faculty of Medicine, Catholic University of the Sacred Heart, Largo F. Vito 1, 00168 Rome, Itaty.
Iacopino
Fortunata
F
Lama
Gina
G
Capucci
Susanna
S
Zelano
Giovanni
G
Boca
Manila
M
Pistilli
Alessandra
A
Sica
Gigliola
G
D018931:Antineoplastic Agents, Hormonal / Q000494:pharmacology*; D017209:Apoptosis / Q000187:drug effects / Q000235:genetics*; D045744:Cell Line, Tumor; D017373:Cyproterone Acetate / Q000494:pharmacology; D013196:Dihydrotestosterone / Q000494:pharmacology; D019254:Genes, bcl-2 / Q000187:drug effects; D016259:Genes, myc / Q000187:drug effects; D007987:Gonadotropin-Releasing Hormone / Q000819:agonists / Q000031:analogs & derivatives; D006801:Humans; D016729:Leuprolide / Q000494:pharmacology*; D008297:Male; D011471:Prostatic Neoplasms / Q000188:drug therapy / Q000235:genetics* / Q000378:metabolism / Q000473:pathology*; D019253:Proto-Oncogene Proteins c-bcl-2 / Q000096:biosynthesis / Q000235:genetics; D016271:Proto-Oncogene Proteins c-myc / Q000096:biosynthesis / Q000235:genetics; D012333:RNA, Messenger / Q000096:biosynthesis / Q000235:genetics; D051028:bcl-2-Associated X Protein
D018931:Antineoplastic Agents, Hormonal; C494826:BAX protein, human; D019253:Proto-Oncogene Proteins c-bcl-2; D016271:Proto-Oncogene Proteins c-myc; D012333:RNA, Messenger; D051028:bcl-2-Associated X Protein; D013196:Dihydrotestosterone; D007987:Gonadotropin-Releasing Hormone; D017373:Cyproterone Acetate; D016729:Leuprolide