Dasatinib enhances tumor growth in gemcitabine-resistant orthotopic bladder cancer xenografts

Abstract

Background: Systemic chemotherapy with gemcitabine and cisplatin is standard of care for patients with metastatic urothelial bladder cancer. However, resistance formation is common after initial response. The protein Src is known as a proto-oncogene, which is overexpressed in various human cancers. Since there are controversial reports about the role of Src in bladder cancer, we evaluated the efficacy of the Src kinase inhibitor dasatinib in the urothelial bladder cancer cell line RT112 and its gemcitabine-resistant sub-line RT112^rGEMCI²⁰ in vitro and in vivo.

Methods: RT112 urothelial cancer cells were adapted to growth in the presence of 20 ng/ml gemcitabine (RT112^rGEMCI²⁰) by continuous cultivation at increasing drug concentrations. Cell viability was determined by MTT assay, cell growth kinetics were determined by cell count, protein levels were measured by western blot, and cell migration was evaluated by scratch assays. In vivo tumor growth was tested in a murine orthotopic xenograft model using bioluminescent imaging.

Results: Dasatinib exerted similar effects on Src signaling in RT112 and RT112^rGEMCI²⁰ cells but RT112^rGEMCI²⁰ cells were less sensitive to dasatinib-induced anti-cancer effects (half maximal inhibitory concentration (IC₅₀) of dasatinib in RT112 cells: 349.2 ± 67.2 nM; IC₅₀ of dasatinib in RT112^rGEMCI²⁰ cells: 1081.1 ± 239.2 nM). Dasatinib inhibited migration of chemo-naive and gemcitabine-resistant cells. Most strikingly, dasatinib treatment reduced RT112 tumor growth and muscle invasion in orthotopic xenografts, while it was associated with increased size and muscle-invasive growth in RT112^rGEMCI²⁰ tumors.

Conclusion: Dasatinib should be considered with care for the treatment of urothelial cancer, in particular for therapy-refractory cases.

Keywords: Acquired resistance; Cancer cell line collection; Dasatinib; Gemcitabine; Orthotopic xenograft model; Urothelial bladder cancer.

Fig. 1

a Growth kinetics of the urothelial cancer cell line RT112 and its gemcitabine-resistant sub-line RT112^rGEMCI²⁰ in cell culture. On day 0, both cell lines started with 4000 cells per milliliter. b In vivo growth kinetics of RT112luc and RT112^rGEMCI²⁰luc tumors in an orthotopic nu/nu mouse xenograft model as determined by bioluminescence. 10 × 10⁸ photons/s correlate to 10 mm³ tumor volume (21). c Concentration-dependent effects of dasatinib on the viability of the urothelial cancer cell line RT112 and its gemcitabine-resistant sub-line RT112^rGEMCI²⁰ as indicated by MTT assay after 120 h of incubation

Fig. 2

a Representative western blots showing cellular levels of Src, pSrc, Akt, pAkt, and β-actin in RT112 and RT112^rGEMCI²⁰ cells after 24 h incubation with dasatinib. b Pixel density of western blots is given in percentage compared to untreated cell line (100 %). One of three independent experiments is shown here. *p ≤ 0.05 vs untreated cell line. c Wound healing migration assay. Effects of dasatinib (100 nM) treatment on RT112 or RT112^rGEMCI²⁰ cells were evaluated. Images of the closure of the scratch were captured at 0, 6, and 24 h. Representative images of scratch wound assays were shown (magnification ×100). d Quantification of wound repair was obtained from six measurements of every treatment from three independent experiments

Fig. 3

Tumor growth kinetics of murine orthotopic a RT112luc and b RT112^rGEMCI²⁰luc xenografts. Dasatinib was given per oral gavage at a dose of 20 mg/kg body weight (BW) twice daily. Control animals were vehicle-treated in the same way. 10 × 10⁸ photons/s correlate to 10 mm³ tumor volume (21). c Representative photographs of treated and untreated RT112luc and RT112^rGEMCI²⁰luc xenografts with fluorescent bladder tumors

Fig. 4

H&E staining of four representative tumors (magnification ×5). a RT112luc xenograft from a vehicle-treated mouse. b RT112luc xenograft from a dasatinib (20 mg/kg BW twice daily)-treated mouse. c RT112^rGEMCI²⁰luc xenograft from a vehicle-treated mouse. d RT112^rGEMCI²⁰luc xenograft from a dasatinib (20 mg/kg BW twice daily)-treated mouse