Korean J Urol.  2011 Jan;52(1):55-63.

Sunitinib Malate Synergistically Potentiates Anti-Tumor Effect of Gemcitabine in Human Bladder Cancer Cells

• Yoon CY
• Lee JS
• Kim BS
• Jeong SJ
• Hong SK
• Byun SS
• Lee SE

Abstract

PURPOSE
Sunitinib malate (Sutent; Pfizer, New York, NY, USA) is a highly selective multi-targeted agent and has been reported to have potent anti-tumor effects against various tumors, including renal cell carcinoma and gastrointestinal stromal tumors. In this study, we explored in vitro the anti-tumor effect and related molecular mechanisms of sunitinib malate against human bladder cancer cell lines. We also determined the synergistic anti-tumor effect between sunitinib and conventional cytotoxic drugs, cisplatin and gemcitabine, in bladder cancer cells.
MATERIALS AND METHODS
Six human cancer cell lines (HTB5, HTB9, T24, UMUC14, SW1710, and J82) were exposed to an escalating dose of sunitinib alone or in combination with cisplatin/gemcitabine, and the cytotoxic effect of the drugs was examined by CCK-8 assay. The synergistic effect between sunitinib and cisplatin/gemcitabine was determined by the combination index (CI) and clonogenic assay. Alterations in cell cycle (cyclin D, B1), survival (p-Akt, t-Akt), and apoptosis (Bax, Bad) regulator expression were analyzed by Western blotting.
RESULTS
Like cisplatin and gemcitabine, sunitinib exerted a dose- and time-dependent anti-tumor effect in bladder cancer cells. However, sunitinib exhibited entirely different sensitivity profiles from cisplatin and gemcitabine. Sunitinib suppressed the expression of cyclin B1, p-Akt, and t-Akt while augmenting the expression of cyclin D and pro-apoptotic Bax and Bad in HTB5 cells. Analysis of the drug combination by the isobolic method and clonogenic assay revealed that sunitinib acts in synergy with gemcitabine in HTB5 cells.
CONCLUSIONS
These results indicate that sunitinib malate has a potent anti-tumor effect and may synergistically enhance the anti-tumor effect of gemcitabine in human bladder cancer cells.

Keyword

Carcinoma; Cisplatin; Gemcitabine; Sunitinib; Urinary bladder

MeSH Terms

Apoptosis
Carcinoma, Renal Cell
Cell Cycle
Cell Line
Cisplatin
Cyclin B1
Cyclin D
Deoxycytidine
Gastrointestinal Stromal Tumors
Humans
Indoles
New York
Pyrroles
Sincalide
Urinary Bladder
Urinary Bladder Neoplasms
Cisplatin
Cyclin B1
Cyclin D
Deoxycytidine
Indoles
Pyrroles
Sincalide

Figure

• 

  FIG. 1 Dose- and time-dependent anti-tumor effect of sunitinib malate in human bladder cancer cell lines. Six human bladder transitional cell carcinoma cell lines of various differentiation (HTB5 - grader 4; HTB9 - grade 2; T24 - grade 3; UMUC14 - grade 4; SW1710 - grade 3; and J82 - grade 3) were exposed to escalating doses of sunitinib malate (0.313-20 µM) for 24, 48, and 72 hours and the anti-tumor effect in each cell line was determined by CCK-8 assay. Each data point represents the Mean±SD of at least three independent experiments.

• 

  FIG. 2 Analysis of of cell cycle and survival regulatory protein expression in the HTB5 cell line. (A) HTB5 cells were exposed to increasing doses of sunitinib malate (5.0 µM, 10.0 µM, and 20.0 µM) for 72 hours and cyclin D, cyclin B1, p-Akt, t-Akt, Bax, and Bad expression was determined by Western blotting. (B) p-Akt and t-Akt expression was measured by densitometric method and percentile changes from untreated control were analyzed. Each data point represents the Mean±SD of two independent experiments. (a and b: p<0.05, respectively; control vs. each treatment).

• 

  FIG. 3 Combination treatment of human bladder cancer cells with sunitinib malate. To check the synergistic anti-tumor effect between sunitinib malate and a conventional chemotherapy agent, the HTB5 cell line was exposed to increasing doses of sunitinib malate alone or in combination with gemcitabine (A) or cisplatin (B) at a fixed ratio (1:1) for 48 hours and the anti-tumor effect was analyzed by CCK-8 assay. The median-effect plot (C) and the dose-effect plot (E) of the sunitinib and gemcitabine combination showed a synergistic anti-tumor effect for the mid-range of dose combinations, whereas the sunitinib and cisplatin combination (D, F) showed no significant synergistic effect over the whole-dose combinations tested.

• 

  FIG. 4 Colony formation of sunitinib-treated HTB5 cells. Two thousand HTB5 cells were seeded in 6 cm culture plates and permitted to attach overnight. After 48 hours of single or combination treatment with sunitinib malate and cisplatin, cells were washed with PBS and cultured in drug-free media for an additional 7 days before crystal violet staining. Only the number of colonies having a diameter of 0.2 mm or larger were counted for analysis. Changes in colony formation in with sunitinib malate ((B) 1.25 µM, (C) 2.5 µM, (D) 5.0 µM) or cisplatin ((E) 0.625 µg/ml) single treatment or combination treatment ((F) 2.5 µM of sunitinib malate plus 0.625 µg/ml of cisplatin) from untreated control (A) are expressed as percentages. Each data point represents the Mean±SD of duplicated experiments.

• 

  FIG. 5 fa-combination index (CI) plot of sunitinib malate combination treatment. HTB5 cells were treated with escalating concentrations of gemcitabine (A) or cisplatin (B) either alone or in combination with sunitinib at a 1:1 fixed ratio for 48 hours and the CI was calculated by using the Chou and Talalay equation as described previously. On the basis of the CI values at each fraction affected (fa), the fa-CI plot was generated in which CI<1, CI=1, and CI>1 denote synergism, additivity, and antagonism, respectively.

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