Effects of alpha-difluoromethylornithine on thrombospondin-1 production by human breast cancer cells.
We have previously observed that inhibition of polyamine biosynthesis with alpha-difluoromethylornithine (DFMO) upregulates production of thrombospondin-1 (TSP-1), an extracellular matrix protein with potent anti-angiogenic and antimetastatic properties, by MDA-MB-435 human breast cancer cells in culture. The present experiments were designed to investigate the mechanisms by which DFMO regulates TSP-1 production in this system. 35S-methionine pulse chase experiments indicated that DFMO administration increased TSP-1 synthesis by approximately 6-fold, while it slightly but significantly decreased protein half-life from 35 to 28 min. DFMO treatment increased steady state TSP-1 mRNA levels by 2-fold in MDA-MB-435 cells. TSP-1 promoter reporter studies indicated that this increase was largely due to activation of transcription. Analysis of distribution of TSP-1 mRNA levels between non-polysomal, subpolysomal and polysomal fractions in control and DFMO-treated cells suggested a major stimulatory effect of the drug on TSP-1 translation. A similar increase in TSP-1 transcription and translation in response to DFMO treatment was also observed in vivo in MDA-MB-435 breast cancer xenografts. Surprisingly however, we failed to detect an increase in TSP-1 protein as assessed by Western blot analysis. The reason for this unexpected finding is unknown but may be due to DFMO-induced stimulation of TSP-1 secretion into the systemic circulation, thus preventing its accumulation within the tumor.
Manni
Andrea
A
Division of Endocrinology, Hershey Medical Center, Pennsylvania State University College of Medicine, Hershey, PA 17033, USA. amanni@psu.edu
Rager
Terry
T
Kimball
Scot R
SR
Jefferson
Leonard S
LS
Washington
Sharlene
S
Hu
Xin
X
Verderame
Michael F
MF
D000818:Animals; D000970:Antineoplastic Agents / Q000494:pharmacology*; D001943:Breast Neoplasms / Q000378:metabolism*; D045744:Cell Line, Tumor; D000518:Eflornithine / Q000494:pharmacology*; D004791:Enzyme Inhibitors / Q000494:pharmacology*; D005260:Female; D006801:Humans; D051379:Mice; D012333:RNA, Messenger / Q000032:analysis; D019700:Thrombospondin 1 / Q000096:biosynthesis* / Q000235:genetics
D000970:Antineoplastic Agents; D004791:Enzyme Inhibitors; D012333:RNA, Messenger; D019700:Thrombospondin 1; D000518:Eflornithine