Induction of apoptosis and enhancement of chemosensitivity in human prostate cancer LNCaP cells using bispecific antisense oligonucleotide targeting Bcl‐2 and Bcl‐xL genes

OBJECTIVE To determine whether a specifically designed bispecific (Bcl‐2/Bcl‐xL) antisense oligonucleotide (ASO) induces apoptosis and enhances chemosensitivity in human prostate cancer LNCaP cells, as Bcl‐2 and Bcl‐xL are both anti‐apoptotic genes associated with treatment resistance and tumour progression in many malignancies, including prostate cancer. MATERIALS AND METHODS Inhibition of Bcl‐2 and Bcl‐xL expression by the bispecific ASO was evaluated using real‐time reverse transcription‐polymerase chain reaction and Western blotting, while growth inhibition and induction of apoptosis were analysed by a crystal violet assay, flow cytometry and Western blotting of apoptosis‐relevant proteins. The effect of combined treatment with bispecific ASO and chemotherapy or small‐interference RNA (siRNA) targeting the clusterin gene was also investigated. RESULTS Bispecific ASO reduced Bcl‐2 and Bcl‐xL expression in LNCaP cells in a dose‐dependent manner. There was cell growth inhibition, increases in the sub‐G0–G1 fraction, and cleavage of caspase‐3 and poly(ADP‐Ribose) polymerase proteins in LNCaP cells after bispecific ASO treatment. Interestingly, Bcl‐2/Bcl‐xL bispecific ASO treatment also resulted in the down‐regulation of Mcl‐1 and up‐regulation of Bax. The sensitivity of LNCaP cells to mitoxantrone, docetaxel or paclitaxel was significantly increased, reducing the 50% inhibitory concentration by 45%, 80% or 90%, respectively. Furthermore, the apoptotic induction by Bcl‐2/Bcl‐xL bispecific ASO was synergistically enhanced by siRNA‐mediated inhibition of clusterin, a cytoprotective chaperone that interacts with and inhibits activated Bax. CONCLUSIONS These findings support the concept of the targeted suppression of Bcl‐2 anti‐apoptotic family members using multitarget inhibition strategies for prostate cancer, through the effective induction of apoptosis.