Combining the receptor tyrosine kinase inhibitor AEE788 and the mammalian target of rapamycin (mTOR) inhibitor RAD001 strongly inhibits adhesion and growth of renal cell carcinoma cells

Abstract

Background: Treatment options for metastatic renal cell carcinoma (RCC) are limited due to resistance to chemo- and radiotherapy. The development of small-molecule multikinase inhibitors has now opened novel treatment options. We evaluated the influence of the receptor tyrosine kinase inhibitor AEE788, applied alone or combined with the mammalian target of rapamycin (mTOR) inhibitor RAD001, on RCC cell adhesion and proliferation in vitro.

Methods: RCC cell lines Caki-1, KTC-26 or A498 were treated with various concentrations of RAD001 or AEE788 and tumor cell proliferation, tumor cell adhesion to vascular endothelial cells or to immobilized extracellular matrix proteins (laminin, collagen, fibronectin) evaluated. The anti-tumoral potential of RAD001 combined with AEE788 was also investigated. Both, asynchronous and synchronized cell cultures were used to subsequently analyze drug induced cell cycle manipulation. Analysis of cell cycle regulating proteins was done by western blotting.

Results: RAD001 or AEE788 reduced adhesion of RCC cell lines to vascular endothelium and diminished RCC cell binding to immobilized laminin or collagen. Both drugs blocked RCC cell growth, impaired cell cycle progression and altered the expression level of the cell cycle regulating proteins cdk2, cdk4, cyclin D1, cyclin E and p27. The combination of AEE788 and RAD001 resulted in more pronounced RCC growth inhibition, greater rates of G0/G1 cells and lower rates of S-phase cells than either agent alone. Cell cycle proteins were much more strongly altered when both drugs were used in combination than with single drug application. The synergistic effects were observed in an asynchronous cell culture model, but were more pronounced in synchronous RCC cell cultures.

Conclusion: Potent anti-tumoral activitites of the multikinase inhibitors AEE788 or RAD001 have been demonstrated. Most importantly, the simultaneous use of both AEE788 and RAD001 offered a distinct combinatorial benefit and thus may provide a therapeutic advantage over either agent employed as a monotherapy for RCC treatment.

Figure 1

Effects of AEE788 (1a) or RAD001 (1b) on kidney cancer proliferation in vitro. A498, Caki-1 or KTC-26 cells were treated with various concentrations of AEE788 or RAD001, or remained untreated (control). Cell proliferation was then assessed using the MTT dye reduction assay. Cell numbers at day 2 and 3 (48 h and 72 h) were compared to the number on day 1 (24 h, as 100%). One representative of 6 experiments is shown. * indicates significant difference to controls (p < 0.05). SD_intraassay < 15%.

Figure 2

Adhesion of RCC cells to HUVEC. A498, Caki-1 or KTC-26 cells were treated with 1 μM AEE788 or 1 nM RAD001, applied alone or in combination. After a 1 h pre-incubation, tumor cells were added at a density of 0.5 × 10⁶ cells/well to HUVEC monolayers for 60 min. Non-adherent tumor cells were washed off in each sample, the remaining cells were fixed and counted in five different fields (5 × 0.25 mm²) using a phase contrast microscope. Mean values were calculated from five counts. Mean adhesion capacity is depicted as adherent cells/mm². One representative of six experiments is shown. * indicates significant difference to controls (p < 0.05).

Figure 3

Adhesion of RCC cells to extracellular matrix proteins. A498, Caki-1 or KTC-26 cells were treated with 1 μM AEE788 or 1 nM RAD001, applied alone or in combination. Non-treated cells served as the controls. Cells were then added to immobilized collagen, laminin, or fibronectin at a density of 0.5 × 10⁶ cells/well for 60 min. Plastic dishes were used to evaluate unspecific binding (background control). Non-adherent tumor cells were washed off in each sample, the remaining cells were fixed and counted in five different fields (5 × 0.25 mm²) using a phase contrast microscope. Mean values were calculated from the five counts. Specific adhesion capacity (background adhesion on plastic surface was subtracted from adhesion to matrix proteins) is depicted as adherent cells/mm². One representative of six experiments is shown. * indicates significant difference to controls (p < 0.05).

Figure 4

Effects of RAD001, AEE788 or erlotinib versus drug combination on kidney cancer proliferation in vitro. A498, Caki-1 or KTC-26 cells were treated with 1 nM RAD001, 1 μM AEE788 or 1 μM erlotinib, applied alone or in combination. Controls remained untreated. Cells were then counted after a further 24, 48 and 72 h using the MTT dye reduction assay. One representative of 6 experiments is shown. * indicates significant difference to controls (p < 0.05).

Figure 5

Effects of RAD001, gefitinib or sunitinib versus drug combination on kidney cancer proliferation in vitro. A498, Caki-1 or KTC-26 cells were treated with 1 nM RAD001, 1 μM gefitinib or 1 μM sunitinib, applied alone or in combination. Controls remained untreated. Cells were then counted after a further 24, 48 and 72 h using the MTT dye reduction assay. One representative of 6 experiments is shown. * indicates significant difference to controls (p < 0.05).

Figure 6

Cell cycle analysis of A498 (6a) and Caki-1 cells (6b). Asynchronous (left) and synchronous (right) cell cultures were used. Cells were treated either with 1 μM or 5 μM AEE788 or with 1 nM or 5 nM RAD001, or with a 1 μM AEE788-1nM RAD001 combination. Controls remained untreated. The cell population at each specific checkpoint is expressed as percentage of the total cells analyzed. One representative experiment of three is shown.

Figure 7

Western blot analysis of cell cycle proteins, listed in methods. Asynchronous A498, Caki-1 or KTC-26 cells were treated either with 1 μM or 5 μM AEE788 or with 1 nM or 5 nM RAD001, or with a 1 μM AEE788-1nM RAD001 combination. Controls remained untreated. Drugs were applied for 6 or 24 h. Cell lysates were then subjected to SDS-PAGE and blotted on the membrane incubated with the respective monoclonal antibodies. Beta-actin served as the internal control. The figure shows one representative from three separate experiments.

Figure 8

Western blot analysis of cell cycle proteins, listed in methods. Synchronized A498 (Fig. 8), Caki-1 (Fig. 9) or KTC-26 cells (Fig. 10) were treated either with 1 μM or 5 μM AEE788 or with 1 nM or 5 nM RAD001, or with a 1 μM AEE788-1nM RAD001 combination. Controls remained untreated. Drugs were applied for 1, 3, 6, or 24 h. Cell lysates were then subjected to SDS-PAGE and blotted on the membrane incubated with the respective monoclonal antibodies. Beta-actin served as the internal control. The figures show one representative from three separate experiments.

Figure 9

Western blot analysis of cell cycle proteins, listed in methods. Synchronized A498 (Fig. 8), Caki-1 (Fig. 9) or KTC-26 cells (Fig. 10) were treated either with 1 μM or 5 μM AEE788 or with 1 nM or 5 nM RAD001, or with a 1 μM AEE788-1nM RAD001 combination. Controls remained untreated. Drugs were applied for 1, 3, 6, or 24 h. Cell lysates were then subjected to SDS-PAGE and blotted on the membrane incubated with the respective monoclonal antibodies. Beta-actin served as the internal control. The figures show one representative from three separate experiments.

Figure 10

Western blot analysis of cell cycle proteins, listed in methods. Synchronized A498 (Fig. 8), Caki-1 (Fig. 9) or KTC-26 cells (Fig. 10) were treated either with 1 μM or 5 μM AEE788 or with 1 nM or 5 nM RAD001, or with a 1 μM AEE788-1nM RAD001 combination. Controls remained untreated. Drugs were applied for 1, 3, 6, or 24 h. Cell lysates were then subjected to SDS-PAGE and blotted on the membrane incubated with the respective monoclonal antibodies. Beta-actin served as the internal control. The figures show one representative from three separate experiments.

Figure 11

Western blot analysis of cell signaling proteins, listed in methods. A498 or Caki-1 cells were treated either with 1 μM AEE788 or with 1 nM RAD001, or with a 1 μM AEE788-1nM RAD001 combination. Controls remained untreated. Drugs were applied for 24 h. Cells were then kept for 2 h in serum-free cell culture medium and subsequently stimulated for 30 min with EGF (100 ng/ml). Cell lysates were subjected to SDS-PAGE and blotted on the membrane incubated with the respective monoclonal antibodies. Beta-actin served as the internal control. The figure shows one representative from three separate experiments.