Small interference RNA targeting heat-shock protein 27 inhibits the growth of prostatic cell lines and induces apoptosis via caspase-3 activation in vitro.
OBJECTIVE
To evaluate synthetic small interference RNA (siRNA) compounds targeting heat-shock protein 27 (Hsp27) as an alternative approach to Hsp27 'knockdown' in prostate cancer cells, as Hsp27 expression is highly up-regulated in prostate cancer cells after androgen withdrawal or chemotherapy, to become uniformly highly expressed in androgen-independent (AI) prostate cancer.


METHODS
We recently showed that targeting Hsp27 by a 2'-methoxyethyl modified phosphorothioate antisense oligonucleotide, OGX-427, inhibits Hsp27 expression and enhances hormone- and chemotherapy in prostate cancer xenograft models. In the present study, a 'gene walk' screening different siRNAs was initially used in PC-3 and LNCaP cells to determine the most potent sequence to down-regulate Hsp27 mRNA and protein levels. The effects of Hsp27 silencing on in vitro growth rates were studied by tetrazolium-blue and crystal violet assays. Apoptosis was determined by single-stranded DNA nuclear and cleaved caspase-3 immunostaining, as well as flow cytometry. Spotted microarrays with 14,000 human oligonucleotides were used to examine changes in gene expression.


RESULTS
Low concentrations of 1 nm siRNA decreased Hsp27 mRNA levels by 19-fold and suppressed protein expression to undetectable levels. Silencing of Hsp27 in prostate cancer cells by siRNA # 2 increased apoptotic rates 2.4-4 fold and caused 40-76% inhibition of cell growth in LNCaP and PC-3 cells. Characteristic cleavage of caspase-3 occurred after treatment with Hsp27 siRNA (1 nm). cDNA microarray analysis from LNCaP and PC-3 cell lines revealed differential gene expression profiles after Hsp27 down-regulation that could be used to identify various survival pathways involved in androgen-dependent and AI growth.


CONCLUSIONS
These findings illustrate the potential utility of Hsp27-silencing therapy and highlight Hsp27 siRNA strategies as a novel and highly effective tool, with the potential for future targeted therapy in enhancing the efficacy of chemotherapy in advanced prostate cancer.
Rocchi
Palma
P
The Prostate Centre, Vancouver General Hospital, Vancouver, British Columbia, Canada.
Jugpal
Paul
P
So
Alan
A
Sinneman
Shannon
S
Ettinger
Susan
S
Fazli
Ladan
L
Nelson
Colleen
C
Gleave
Martin
M
D017209:Apoptosis / Q000187:drug effects; D015152:Blotting, Northern; D015153:Blotting, Western; D053148:Caspase 3 / Q000378:metabolism*; D045744:Cell Line, Tumor; D015536:Down-Regulation / Q000187:drug effects; D004789:Enzyme Activation; D005434:Flow Cytometry; D006360:Heat-Shock Proteins / Q000187:drug effects* / Q000378:metabolism; D006801:Humans; D008297:Male; D020411:Oligonucleotide Array Sequence Analysis; D011471:Prostatic Neoplasms / Q000188:drug therapy*; D034741:RNA, Small Interfering / Q000494:pharmacology* / Q000627:therapeutic use; D014407:Tumor Cells, Cultured; D015854:Up-Regulation / Q000187:drug effects
D006360:Heat-Shock Proteins; D034741:RNA, Small Interfering; D053148:Caspase 3