Ribozyme-targeting procathepsin D and its effect on invasion and growth of breast cancer cells: an implication in breast cancer therapy.

Abstract	Procathepsin D (pCD), a zymogen of lysosomal aspartic peptidase cathepsin D, overexpression is correlated with highly invasive malignancies, including breast cancer. Recently, different studies have shown the role of secreted pCD as mitogen acting both in an autocrine and a paracrine manner. The aim of the present study is to examine the anti-tumor effects elicited by a decrease in the protein level of pCD by ribozyme and to explore the therapeutic potential of this specific targeting. Using the mFold program, we designed seven anti-pCD ribozymes and checked the accessibility to target pCD mRNA by RNase H cleavage experiment in a cell-free system. The sequences of the 4 most effective ribozymes were cloned and stably transfected in a highly metastatic human breast cancer cell line, MDA-MB-231, to knock down the expression of pCD. Downregulation of pCD due to ribozyme expression was observed by Western blotting and real-time RT-PCR. Stably transfected cells with anti-pCD ribozymes exhibited a significant lowering of in vitro invasion (p<0.001) and reduction in lung colonization potential in nude mice when compared to control ribozyme transfected cells. We also found that downregulation of pCD by ribozyme promotes apoptosis of MDA-MB-231 cells on serum deprivation. These results suggest that we have generated a biologically functional ribozyme against pCD with possible therapeutic implications in breast cancer cells.
Authors	Aruna Vashishta, Sujata Saraswat Ohri, Mary Proctor, Martin Fusek, Vaclav Vetvicka
Journal	International journal of oncology (Int J Oncol) Vol. 30 Issue 5 Pg. 1223-30 (May 2007) ISSN: 1019-6439 [Print] Greece
PMID	17390025 (Publication Type: Journal Article, Research Support, N.I.H., Extramural)
Chemical References	Antineoplastic Agents Enzyme Precursors RNA, Catalytic procathepsin D Cathepsin D
Topics	Animals Antineoplastic Agents (pharmacology) Breast Neoplasms (drug therapy, metabolism) Cathepsin D (metabolism) Cell Line, Tumor Cell Proliferation Enzyme Precursors (metabolism) Gene Expression Regulation, Neoplastic Humans Mice Mice, Nude Neoplasm Metastasis Neoplasm Transplantation RNA, Catalytic (chemistry, pharmacology) Time Factors Transfection

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