In vitro and in vivo radiosensitization with AZD6244 (ARRY-142886), an inhibitor of mitogen-activated protein kinase/extracellular signal-regulated kinase 1/2 kinase

Abstract

Purpose: The mitogen-activated protein (MAP) kinase pathway is important for cell proliferation, survival, and differentiation, and is frequently up-regulated in cancers. The MAP kinase pathway is also activated after exposure to ionizing radiation. We investigated the effects of AZD6244 (ARRY-142886), an inhibitor of MAP kinase/extracellular signal-regulated kinase 1/2, on radiation response.

Experimental design: The effects of AZD6244 on the in vitro radiosensitivity of human cancer cell lines (A549, MiaPaCa2, and DU145) were evaluated using clonogenic assays. DNA damage repair was evaluated using gammaH2AX, and mitotic catastrophe was measured using nuclear fragmentation. Cell cycle effects were measured with flow cytometry. Growth delay was used to evaluate the effects of AZD6244 on in vivo tumor radiosensitivity.

Results: Exposure of each cell line to AZD6244 before irradiation resulted in an increase in radiosensitivity with dose enhancement factors at a surviving fraction of 0.1, ranging from 1.16 to 2.0. No effects of AZD6244 on radiation-induced apoptosis or persistence of gammaH2AX foci after irradiation were detected. Cells treated with AZD6244 had an increased mitotic index and decreased Chk1 phosphorylation at 1 and 2 hours after irradiation. Mitotic catastrophe was increased in cells receiving AZD6244 and irradiation compared with the single treatments. In vivo studies revealed that AZD6244 administration to mice bearing A549 tumor xenografts resulted in a greater than additive increase in radiation-induced tumor growth delay (dose enhancement factor of 3.38).

Conclusions: These results indicate that AZD6244 can enhance tumor cell radiosensitivity in vitro and in vivo and suggest that this effect involves an increase in mitotic catastrophe.

Figure 1. The effects of AZD6244 on tumor cell radiosensitivity

Cell lines A549 (A), MiaPaCa2 (B), and DU145 (C) were exposed to AZD6244 (250 nM for A549 and DU145, 100 nM for MiaPaCa2) or vehicle control for 16 hours, and irradiated with graded doses of X-rays. Colony-forming efficiency was determined 10 to 14 days later and survival curves generated after normalizing for cell killing by AZD6244 alone. The data represent the mean of three independent experiments. PE, plating efficiency with AZD6244; DEF, dose enhancement factor. Points, mean; bars, ±SE.

Figure 2. The effects of AZD6244 on ERK1/2 phosphorylation

The effects of AZD6244 on ERK1/2 phosphorylation. The A549 (A), MiaPaCa2 (B), and DU145 (C) cell lines were exposed to AZD6244 (250 nM for A549 and DU145, 100 nM for MiaPaCa2) or vehicle control for 16 hours, irradiated, harvested, and subjected to immunoblotting and densitometry. ERK1/2 phosphorylation was induced by radiation and reduced following exposure to AZD6244. C, control untreated cells; AZD, cells exposed to AZD6244 for 16 hours prior to harvesting or IR; 1h, 2h, 6h, time after exposure to IR. Densitometry was done using ImageQuant 5.2 software.

Figure 3. The effects of AZD 6244 on the cell cycle

A, A549 cells were exposed to 250 nM AZD6244 or vehicle control for 16 hours prior to evaluation of cell cycle distribution with flow cytometry. *, P < 0.01 according to student’s t test (vehicle treatment vs. AZD6244 treatment). B, A549 cells were stained with propidium iodide at the specified times and analyzed using flow cytometry. To evaluate the activation of G2 cell cycle checkpoint, mitotic cells were distinguished from G2 cells, and the mitotic index was determined according to the expression of phosphorylated H3 detected in the 4N DNA content population by the flow cytometry. Loss of mitotic cells reflects the onset of G2 arrest. AZD6244, cells that had received AZD6244 (250 nM) alone. Columns, mean; bars, SE.

Figure 4. The effects of AZD6244 on Chk1 phosphorylation

A, A549 cells were exposed to AZD6244 (250 nM) or vehicle control for 16 hours, irradiated, and harvested, and subjected to immunoblotting. Chk1 phosphorylation was induced by radiation and reduced following exposure to AZD6244. C, control untreated cells; AZD, cells exposed to AZD6244 for 16 hours prior to harvesting or IR; 1h, 2h, time after exposure to IR. B, Densitometry was done using by ImageQuant 5.2 software.

Figure 5. The effects of AZD6244 on the mechanism of cell death after irradiation

Apoptosis: Cells were treated with vehicle control or AZD6244 (250 nM) and harvested at the specified times. Treated cell samples were added to a 150 µL staining solution (Guava Nexin Assay) containing 135 µL 1x apoptosis buffer, 10 µL Annexin V-PE, and 5 µL of 7-AAD. Samples (2,000 cells per sample) were evaluated by flow cytometry, A549 (A), MiaPaCa2 (B). Columns, mean; bars, SE. Mitotic Catastrophe: Cells growing in chamber slides were exposed to AZD6244 (250 nM) or vehicle control, irradiated (4 Gy), and fixed at the specified times for immunocytochemical analysis for mitotic catastrophe, A549 (C), MiaPaCa2 (D). Nuclear fragmentation was evaluated in 150 cells per treatment per experiment. Nuclear fragmentation was defined as the presence of two or more distinct lobes within a single cell. Columns, mean; bars, SE. Nuclear fragmentation was defined as the presence of two or more distinct lobes within a single cell. *, P < 0.01 according to student’s t test (IR vs. AZD6244 + IR).

Figure 6. The effects of AZD6244 on A549 and MiaPaCa2 xenograft tumors

When A549 (A) and MiaPaCa2 (B) tumors reached 177 mm³ in size, mice were randomized into four groups: vehicle, AZD6244, radiation (3 Gy), or AZD6244 plus radiation. AZD6244 was given by mouth (oral gavage) in a single dose of 50 mg/kg. Radiation (3 Gy) was delivered 4 hours after AZD6244 treatment. Each treated group contained 5 mice with the vehicle control group containing 10 mice. Points, mean; bars, SE.