Article

Downmodulation of Bcl-2 sensitizes metastatic LNCaP-LN3 cells to undergo apoptosis via the intrinsic pathway.

Department of Urology, University of Texas M. D. Anderson Cancer Center, Houston, Texas 77030, USA.
The Prostate (impact factor: 3.48). 11/2009; 70(6):571-83. DOI:10.1002/pros.21091
Source: PubMed

ABSTRACT We explored the mechanisms of apoptosis after Bcl-2 protein downmodulation in metastatic LNCaP-LN3 cells (LN3).
LNCaP, LNCaP-Pro5 (Pro5) and LN3 cells were cultured in 5% charcoal-stripped serum (CSS) or in R1881 (synthetic androgen) and bicalutamide (synthetic anti-androgen) and growth inhibition was assessed. Expression levels of androgen receptor (AR) and Bcl-2 were determined. LN3 cells were transfected with small interfering RNA Bcl-2 (siRNA Bcl-2) or control siRNA oligonucleotides. Rates of apoptosis and proliferation were obtained. Cytochrome c localization in treated and control cells was assessed +/- cyclosporine A (CsA). Caspases 9, 3, and poly (ADP-ribose) polymerase cleavage (PARP) were measured upon downmodulation of Bcl-2; and cell growth inhibition in vitro after Bcl-2 modulation combined with docetaxel chemotherapy was determined.
LN3 cells maintained growth under castrate conditions in vitro. AR protein amplification did not explain castrate-resistant LN3 cell growth. Bcl-2 protein levels in LN3 cells were significantly higher than in Pro5 cells, and were effectively downmodulated by siRNA Bcl-2. Subsequently increased apoptosis and decreased proliferation mediated by cytochrome c was noted and this was reversed by CsA. siRNA Bcl-2-transfected LN3 cells exhibited elevated levels of caspases 9, 3, and PARP cleavage. Exposure of LN3 cells to docetaxel led to increased apoptosis, and simultaneous downmodulation of Bcl-2 substantially enhanced this effect.
Downmodulation of Bcl-2 in metastatic castrate-resistant LNCaP-LN3 cells led to apoptosis via a cytochrome c-dependent pathway that was enhanced with docetaxel treatment.

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Keywords

+/- cyclosporine
 
5% charcoal-stripped serum
 
AR protein amplification
 
Bcl-2 protein downmodulation
 
Bcl-2 protein levels
 
castrate-resistant LN3 cell growth
 
control cells
 
control siRNA oligonucleotides
 
cytochrome c
 
Cytochrome c localization
 
docetaxel treatment
 
Expression levels
 
LN3 cells
 
metastatic castrate-resistant LNCaP-LN3 cells
 
metastatic LNCaP-LN3 cells
 
Pro5 cells
 
RNA Bcl-2
 
siRNA Bcl-2
 
synthetic androgen
 
synthetic anti-androgen