Ribozyme-targeting procathepsin D and its effect on invasion and growth of breast cancer cells: an implication in breast cancer therapy.
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.
Vashishta
Aruna
A
Department of Pathology, University of Louisville, Louisville, KY 40292, USA.
Ohri
Sujata Saraswat
SS
Proctor
Mary
M
Fusek
Martin
M
Vetvicka
Vaclav
V
D000818:Animals; D000970:Antineoplastic Agents / Q000494:pharmacology*; D001943:Breast Neoplasms / Q000188:drug therapy* / Q000378:metabolism*; D002402:Cathepsin D / Q000378:metabolism*; D045744:Cell Line, Tumor; D049109:Cell Proliferation; D004792:Enzyme Precursors / Q000378:metabolism*; D015972:Gene Expression Regulation, Neoplastic; D006801:Humans; D051379:Mice; D008819:Mice, Nude; D009362:Neoplasm Metastasis; D009368:Neoplasm Transplantation; D016337:RNA, Catalytic / Q000737:chemistry / Q000494:pharmacology*; D013997:Time Factors; D014162:Transfection
D000970:Antineoplastic Agents; D004792:Enzyme Precursors; D016337:RNA, Catalytic; C058345:procathepsin D; D002402:Cathepsin D