PDF - 1214KB, A, About 103 HeLa were seeded in 6 well plates exposed to tiplaxtinin at the indicated concentration for 72 hrs followed by aspiration of culture media and replacement with fresh complete media. HeLa clones (HeLa-PAI-1OE-12, HeLa-PAI-1OE-12 and HeLaEmpty control) overexpressing PAI-1 were also plated similarly. Cells were incubated for an additional 14 days. After 14 days, colonies were fixed with 6.0% glutaraldehyde, stained with 0.5% crystal violet and counted under light microscopy. Data were represented as mean + SD of three independent replicates. B, About 2 x 103 HeLa cells pretreated with tiplaxtinin at the indicated concentration and HeLa clones (HeLa-PAI-1OE-12, HeLa-PAI-1OE-12 and HeLaEmpty control) were mixed in agar solution and fresh complete media and seeded in 96-well plates. Cells were incubated for 6-8 days. Colony formation was quantified using the fluorescent cell stain CyQuant GR Dye (Cell Biolabs Inc., San Diego, CA) in the FLUOstar Optima Reader. Data were represented as mean + SD of three independent replicates. All experiments were repeated at least three times. Targeting PAI-1 was noted to retard cellular proliferation and colony formation. *, p < 0.05
ARTICLE ABSTRACT
Cancers of the urinary bladder result in aggressive and highly angiogenic tumors for which standard treatments have only limited success. Patients with advanced disease have a 5-year survival rate of less than 20%, and no new anticancer agent has been successfully introduced into the clinic armamentarium for the treatment of bladder cancer in more than 20 years. Investigations have identified plasminogen activator inhibitor-1 (PAI-1), a serine protease inhibitor, as being highly expressed in several malignancies, including bladder cancer, in which high expression is associated with a poor prognosis. In this study, we evaluated PAI-1 as a potential therapeutic target for bladder cancer. PAI-1 expression was manipulated in a panel of cell lines and functional inhibition was achieved using the small molecule tiplaxtinin. Reduction or inhibition of PAI-1 resulted in the reduction of cellular proliferation, cell adhesion, and colony formation, and the induction of apoptosis and anoikis in vitro. Treatment of T24 xenografts with tiplaxtinin resulted in inhibition of angiogenesis and induction of apoptosis, leading to a significant reduction in tumor growth. Similar results were obtained through evaluation of the human cervical cancer HeLa cell line, showing that PAI-1–mediated effects are not restricted to tumor cells of bladder origin. Collectively, these data show that targeting PAI-1 may be beneficial and support the notion that novel drugs such as tiplaxtinin could be investigated as anticancer agents. Mol Cancer Ther; 12(12); 2697–708. ©2013 AACR.
