In Vitro and In Vivo Effects of the Urokinase Plasminogen Activator Inhibitor WX-340 on Anaplastic Thyroid Cancer Cell Lines

Abstract

Increased expression of the urokinase-type plasminogen activator (uPA) system is associated with tumor invasion, neo-angiogenesis, and metastatic spread, and has been shown to positively correlate with a poor prognosis in several cancer types, including thyroid carcinomas. In recent years, several uPA inhibitors were found to have anticancer effects in preclinical studies and in some phase II clinical trials, which prompted us to evaluate uPA as a potential therapeutic target for the treatment of patients affected by the most aggressive form of thyroid cancer, the anaplastic thyroid carcinoma (ATC). In this study, we evaluated the in vitro and in vivo effects of WX-340, a highly specific and selective uPA inhibitor, on two ATC-derived cell lines, CAL-62 and BHT-101. The results obtained indicated that WX-340 was able to reduce cell adhesion and invasiveness in a dose-dependent manner in both cell lines. In addition, WX-340 increased uPA receptor (uPAR) protein levels without affecting its plasma membrane concentration. However, this compound was unable to significantly reduce ATC growth in a xenograft model, indicating that uPA inhibition alone may not have the expected therapeutic effects.

Keywords: BHT-101; CAL-62; WX-340; anaplastic thyroid cancer; cell adhesion; cell migration; proliferation; urokinase plasminogen activator; xenograft.

Figure 1

uPA inhibition assay. The activity of 10 Units of uPA purified from human urine was measured with increasing concentrations of WX-340 (50 pM to 50 μM). IC50: half-maximal inhibitory concentration.

Figure 2

Dose-response proliferation assay. Cells were treated with increasing doses of WX-340 for 3 days, after which the tetrazolium salt WST-1 was added to cultures, and production of formazan dye was read after 2 h by microplate reader. The average absorbance value of each point at T = 72 h was subtracted to the average value measured in a plate at T = 0 h.

Figure 3

Adhesion assay. Cells pre-treated with increasing doses of WX-340 for 24 h were detached with trypsin/EDTA, counted, and seeded in a 96-well plate. Separate well rows were washed after 15 min, 30 min, 60 min, or not washed. Finally, WST-1 was added to all wells and the OD was measured 2 h later. * p < 0.05.

Figure 4

Invasion assay through Matrigel-coated nucleopore membranes. Cells pre-treated or not with WX-340 5 μM for 24 h were counted and suspended in serum-free medium with or without fresh added WX-340, then seeded in the upper reservoirs of Boyden chambers. The lower reservoirs were filled with medium containing FBS as chemoattractant. After 6 h incubation, nucleopore membranes were fixed and stained with violet Cresyl solution. Non-migrated cells were removed from the upper layer, and migrated cells were estimated by cell count (CAL-62) or by measuring the ratio black pixels/white pixels in binarized photos of membranes with the ImageJ software 1.53 (BHT-101). * p < 0.05.

Figure 5

(A) Representative Western blotting experiment with BHT-101 treated with increasing doses of WX-340; (B) densitometric analysis of the immunoblotted bands. * p < 0.05; ** p < 0.01.

Figure 6

Citofluorimetric detection of membrane uPAR in BHT-101 cells treated with WX-340 compared to control cells.

Figure 7

(A) Growth curves of tumor masses in ATC xenograft models. For convenience, only one group of treated mice is represented together with the control group. Quantitative analysis of necrosis (B), proliferation index (C), and vessel density (D) in tumor sections of treated mice vs. control mice. Results are expressed as mean ± SD.