In vitro suppression of urokinase plasminogen activator in breast cancer cells--a comparison of two antisense strategies.
High level expression of urokinase plasminogen activator (uPA) has been well documented in a variety of tumors. In breast cancer, expression of uPA is essential for tumor cell invasion, metastasis and proliferation. By contrast, the primary objective of tumor therapy is to reduce the uPA expression level within the tumor, which results in abrogation of proliferation, invasion and metastasizing of the tumor cells. We investigated the effects of uPA on the MDA-MB-231 cell line. MDA-MB-231 cells are highly invasive and express high levels of uPA. In our study, uPA inhibition was achieved by two methodologies: a) stable transfection with an antisense uPA vector, b) transfection with siRNA molecules (small interfering RNA). A plasmid vector was constructed by cloning a uPA-specific cDNA (612 bp) fragment into pBK-CMV plasmid in antisense orientation. In contrast, a double-stranded 21-mer siRNA was designed for targeting uPA. The antisense-transfected cells revealed decreased uPA mRNA and protein as detected by real-time PCR, immunocytochemistry, ELISA, and Western blotting. Moreover, the transfected cells exhibited a significantly reduced proliferation activity as determined by a fluorometric proliferation assay. As a conclusion of our study siRNA-technique is the superior method also regarding time saving for clone selection and instant availability of the transfected cells. Moreover, even if both strategies lead to uPA suppression, a stronger inhibitory effect could be obtained by application of the siRNA-based technique.
Arens
Norbert
N
Universitätsklinikum Mannheim, Pathologisches Institut, D-68167 Mannheim, Germany. norbert.arens@path.ma.uni-heidelberg.de
Gandhari
Mukesh
M
Bleyl
Uwe
U
Hildenbrand
Ralf
R
D001943:Breast Neoplasms / Q000235:genetics / Q000378:metabolism / Q000628:therapy*; D049109:Cell Proliferation / Q000187:drug effects; D016373:DNA, Antisense / Q000235:genetics*; D005260:Female; D005822:Genetic Vectors / Q000235:genetics; D006801:Humans; D034622:RNA Interference; D034741:RNA, Small Interfering / Q000235:genetics*; D014162:Transfection / Q000379:methods; D014568:Urokinase-Type Plasminogen Activator / Q000032:analysis / Q000037:antagonists & inhibitors* / Q000235:genetics
D016373:DNA, Antisense; D034741:RNA, Small Interfering; D014568:Urokinase-Type Plasminogen Activator