Cathepsin D, a lysosomal aspartic proteinase, is overexpressed and secreted as procathepsin D (pCD) by human breast and other cancer cell lines. Studies have shown that procathepsin D has been implicated to play a pivotal role in breast cancer development and progression. This study aim to inhibit pCD synthesis and secretion using specifically designed ribozymes and to evaluate its impact on the proliferation and in vitro invasion of breast cancer cells. Seven hammerhead ribozymes were designed to cleave pCD GUC-recognition site using MFOLD program. The accessibility of the native target mRNA was probed with the antisense oligonucleotides (ODN) using RNase H cleavage assay in cell extract. The sequence of the four effective ribozymes was cloned in pHβApr-1-neo vector. Breast cancer cell line MDA-MB-231 was transfected with the ribozyme-containing plasmids, control ribozyme containing plasmid and vector only. Expression of pCD mRNA and protein was determined. Proliferation and in vitro invasiveness of transfected cells were also analyzed. Stable transfectants of ribozyme-containing plasmids manifested as almost complete loss of pCD mRNA and protein as shown by RT-PCR and Western blotting, respectively while the control ribozyme and wild-type plasmid had no effects. Ribozyme transfected cells exhibited reduced proliferation and in vitro invasiveness through extracellular matrix (Matrigel), compared with the MDA-MB-231 cells and cells transfected with disabled ribozyme and plasmid only. Our data suggest that anti-pCD ribozymes are a potent inhibitor of pCD expression and is an effective approach in reducing cell proliferation and invasiveness of breast cancer cells.

[Proc Amer Assoc Cancer Res, Volume 46, 2005]