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Apoptosis-related gene expression affected by a GnRH analogue without induction of programmed cell death in LNCaP
Cristiana Angelucci1, Fortunata Iacopino, Gina Lama
1Institute of Histology and Embryology, Faculty of Medicine, Catholic University of the Sacred Heart, Largo F. Vito 1, 00168 Rome, Itaty.
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.
Materials And 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.
Conclusion:
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.
Insights
Leuprolide acetate (LA), a GnRH agonist, counteracts 5alpha-dihydrotestosterone (DHT)-stimulated prostate cancer cell growth by modulating gene expression. While LA did not induce apoptosis, it altered key genes, suggesting an antiproliferative mechanism.
Area of Science:
- Oncology
- Molecular Biology
- Endocrinology
Background:
- Androgen-sensitive prostate cancer (LNCaP) cells are stimulated by 5alpha-dihydrotestosterone (DHT).
- Leuprolide acetate (LA), a Gonadotropin Releasing Hormone (GnRH) agonist, has shown potential in suppressing DHT-stimulated prostate cancer cell growth.
- The precise cellular mechanisms underlying LA's effect on gene expression and apoptosis in prostate cancer remain incompletely understood.
Purpose of the Study:
- To investigate the effects of LA, alone and in combination with DHT or cyproterone acetate (CA), on the expression of key genes (bcl-2, bax, c-myc) in LNCaP cells.
- To evaluate the impact of LA on apoptosis in androgen-stimulated prostate cancer cells.
Main Methods:
- Gene expression analysis using RT-PCR and Western blotting for bcl-2, bax, and c-myc.
- Treatment of LNCaP cells with varying concentrations of LA, DHT, and CA.
- Assessment of apoptosis via DNA fragmentation analysis following hormonal treatments.
Main Results:
- DHT upregulated anti-apoptotic bcl-2 and pro-proliferative c-myc, while downregulating pro-apoptotic bax.
- LA counteracted DHT's effects, decreasing bcl-2 and c-myc mRNA/protein and increasing bax protein.
- Cyproterone acetate (CA) mimicked DHT's effects; no significant DNA fragmentation (apoptosis) was observed with LA treatment.
Conclusions:
- LA modulates the expression of genes involved in cell proliferation and apoptosis in prostate cancer cells.
- Despite gene expression changes, LA alone did not induce apoptosis in LNCaP cells.
- The antiproliferative effect of LA on androgen-stimulated prostate cancer cells is likely mediated through these observed gene expression alterations.
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