Pazopanib synergizes with docetaxel in the treatment of bladder cancer cells

Abstract

Objectives: To investigate the efficacy of pazopanib, both alone and in combination with docetaxel, in bladder cancer cells. Bladder cancer expresses many potential therapeutic targets of biological agents, including the vascular endothelial growth factor receptor (VEGFR). Pazopanib is a small molecule inhibitor of VEGFR-1, -2-3, platelet-derived growth factor receptor (PDGFR), and c-Kit.

Materials and methods: Using human bladder cancer cells HTB3, HT1376, J82, RT4, CRL1749, T24, Sup, and HTB9, the treatment effect of pazopanib and cytotoxic chemotherapy was assessed using a tetrazolium-based assay. The combinatorial effect of these agents on clonogenic growth was further examined. Western blotting was used to assess changes in relevant downstream targets, including phospho-AKT, phospho-FAK, total AKT, and total FAK.

Results: Single-agent pazopanib had modest activity. However, synergy was seen with the combination of docetaxel and pazopanib in these multiple cells lines. J82 and T24 cells were selected for additional clonogenic testing because of their resistance to single-agent docetaxel chemotherapy. 1.25 nM of docetaxel had little effect on clonogenic formation; however, in combination with pazopanib, significant inhibition of colony formation was observed. This combination treatment additionally decreased phospho-AKT, an important mediator of cell survival in all cell lines, whereas phospho-FAK expression was variably affected.

Conclusions: The present study demonstrates synergistic efficacy of pazopanib with docetaxel in docetaxel-resistant bladder cancer cells. This work supports future evaluation of pazopanib with docetaxel for the treatment of bladder cancer with the potential of improved efficacy and toxicity.

Figure 1

The treatment effect of pazopanib and docetaxel on bladder cancer cell viability was assessed. Human bladder cancer cells HTB3, HT1376, J82, RT4, CRL1749, T24, SUP and HTB9 were seeded in triplicate with treatment started 24 hours after seeding and continued for 96 hours. Cell viability was assessed with a tetrazolium-based assay. The cells were treated with pazopanib (A) and docetaxel (B) with the concentrations indicated. Bars indicate the standard deviation. All cell lines studied, except RT4, had an inhibitor concentration 50 (IC50) of more than of 10 µM of pazopanib (A). Treatment with docetaxel demonstrated better single-agent activity compared with pazopanib, although J82 cells displayed strong resistance to docetaxel (B); The effect of combination treatment with pazopanib and docetaxel was also evaluated (C–E). Twenty-four hours after plating, the cells were treated by pazopanib and docetaxel concurrently for 96 hours. The cells were J82 (C), T24 (D) and HT1376 (E), with bars indicating the standard deviation. The combination index (CI) levels for concurrent treatment with pazopanib and docetaxel were calculated for J82 (F), T24 (G) and HT1376 (H) cells. CI values of less than 1 are consistent with synergy.

Figure 2

The effect of pazopanib and docetaxel on colony formation in J82 (A) and T24 (B) cells was assessed. The treatment conditions are noted for each well. After 7–10 days of treatment, the cells were stained with Brilliant Blue R. The numbers adjacent to the corresponding wells indicate the mean number ± SD of the colonies per well in each treatment condition.

Figure 3

Expressions of phospho-AKT, phospho-FAK, total AKT, total FAK were determined by Western blot analysis. The cells were J82 (A), T24 (B) and HT1376 (C) respectively. The relative expression ratios of the protein of interest and β-actin are indicated in the figure.