PDF - 1338KB, A, Annexin V apoptotic assay was performed in parental HeLa cells treated with and without tiplaxtinin (30 and 50 microM) and exposed to the apoptotic inducing agent, mitomycin C at 5 microg/ml. The anti-apoptotic effects of PAI-1 overexpression were abrogated in all cells treated with tiplaxtinin. B, Annexin V apoptotic assay was performed in HeLa clones (HeLa-PAI-1OE and HeLaEmpty) after exposing the cells to the apoptotic inducing agent, mitomycin C at 5 ?g/ml. Overexpression of PAI-1 in HeLa clones elicited protection against mitomycin C induced cell death (HeLa-PAI-1KD-12 and HeLa-PAI-1KD-18). C, Western blot analysis was performed on the above cell lines confirming the induction of the key apoptotic proteins cleaved caspase-3, cleaved-PARP as well as Fas and FasL. Staining for β-actin served as a control. At least three independent experiments consisting of each condition tested in triplicate wells was used to calculate mean + SD values. *, p < 0.05. D, In a cell adhesion assay, treatment of HeLa cells with tiplaxtinin resulted in a dose-dependent reduction in the ability of the cells to adhere to substrate. E, In an anoikis assay, treatment of HeLa cells with tiplaxtinin for 24 hrs resulted in significant induction of anoikis.
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.
