Reversal of cathepsin D routing modulation in MCF-7 breast cancer cells expressing antisense insulin-like growth factor II (IGF-II).
We previously demonstrated that expression of IGF-II modulates the routing of cathepsin D in MCF-7 cells. In our present study, we transfected antisense IGF-II into IGF-II secreting MCF-7 cells to test the hypothesis that blocking IGF-II may reduce the secretion of cathepsin D in breast cancer cells. The concentration of IGF-II in media conditioned by the antisense clone was reduced to almost undetectable levels. Likewise, Northern blotting analysis revealed that IGF-II mRNA was nearly undetectable in the antisense transfected cells. Metabolic labeling experiments performed with 10 mM mannose 6-phosphate present in the medium to block reuptake of lysosomal enzymes demonstrated that cathepsin D secretion was dramatically reduced. Similarly, a significant reduction in cathepsin D was observed when conditioned media and cell extracts were examined by Western blotting after a 48 h incubation. No changes in cathepsin D mRNA in antisense cells were detected by Northern blot analysis. We conclude that endogenous IGF-II may modulate the routing of cathepsin D by interfering with receptor trafficking in MCF-7 cells, and that this modulation is reversible. Abnormally high levels of IGF-II may alter this homeostasis, conferring on breast cancer cells an advantageous mechanism that promotes rapid growth, and may facilitate metastasis.
De León
D D
DD
Loma Linda University School of Medicine, CA, USA. ddeleon@som.llu.edu
Issa
N
N
Nainani
S
S
Asmerom
Y
Y
D016375:Antisense Elements (Genetics); D015152:Blotting, Northern; D015153:Blotting, Western; D001943:Breast Neoplasms; D002402:Cathepsin D / Q000032:analysis / Q000235:genetics* / Q000378:metabolism; D015972:Gene Expression Regulation, Neoplastic; D006801:Humans; D007334:Insulin-Like Growth Factor I / Q000235:genetics; D007335:Insulin-Like Growth Factor II / Q000032:analysis / Q000235:genetics* / Q000378:metabolism; D011233:Precipitin Tests; D012333:RNA, Messenger / Q000032:analysis; D017527:Receptor, IGF Type 2 / Q000378:metabolism; D014162:Transfection; D014407:Tumor Cells, Cultured / Q000737:chemistry / Q000378:metabolism
D016375:Antisense Elements (Genetics); D012333:RNA, Messenger; D017527:Receptor, IGF Type 2; D007334:Insulin-Like Growth Factor I; D007335:Insulin-Like Growth Factor II; D002402:Cathepsin D