Figure 1.
Response to AZD6244 or MK2206 alone in various lung cancer cell lines.
The indicated cell lines were treated with different concentrations of either AZD6244 or MK2206 for 96 h. Cell viability was determined using sulforhodamine B, and IC50 was calculated according to the dose-response curve.
Table 1.
Correlations between the IC50 to AZD6244 and gene mutations in lung cancer cell lines.
Table 2.
Correlations between the IC50 to MK2206 and gene mutations in lung cancer cell lines.
Figure 2.
Dose-effect curves for the AZD6244-MK2206 combination for lung cancer cell lines.
Lung cancer cell lines were treated with various concentrations of AZD6244 (0.024–100 µM), MK2206 (0.005–20 µM) and AZD6244/MK2206 (0.024/0.005–100/20 µM) for 96 h. Dose-effect curves of the combination and of each agent alone are presented for comparison. Representative cell lines that demonstrated the strong synergistic effects of the combination therapy are shown.
Table 3.
Combination index (CI) value of the combination therapy of AZD6244 and MK2206 at the ratio of 5∶1.
Figure 3.
Treatment with AZD6244 and MK2206 synergistically upregulated Bim expression and induced cell apoptosis.
(A) A549 and H157 were treated with AZD6244, MK2206, or a combination of these two compounds at the indicated concentrations for 48 h. (B) Protein specimens were harvested and AKT, p-AKT, ERK, p-ERK and Bim expression were detected with Western blot analysis. (C) Cells were trypsinized and fixed, and the cell cycle was measured with flow cytometry. Numbers represent percentages of apoptotic sub-G1–phase cells. Data represent one of three independent experiments with similar results. Columns, mean; bar, SD.*, P<0.05, compared with the single agent treatments.
Table 4.
Combination index (CI) value of the combination therapy of AZD6244 and MK2206 at the ratios of 8∶1, 4∶1, 2∶1, and 1∶8.
Figure 4.
Effect of AZD6244-MK2206 combination on tumor growth and survival in mice.
Flank tumors were established in nude mice and treated with vehicle, AZD6244, MK2206, or combination AZD6244-MK2206 therapy orally twice a day at indicated doses. (A) Tumor volumes. Overall differences between treatments were statistically significant (P<0.001; Kruskal-Wallis Test). We observed statistically significant time-by-treatment effects when comparing Control vs A (P<0.05); Control vs M (P<0.05); Control vs A+M (P<0.05); A vs M (P = 0.05); A vs A+M (P = 0.05) and M vs A+M (P<0.05) for A549; results were similar for H157 except that A vs. M was not statistically significant. (B) Survival times. Overall differences between treatments were statistically significant (P<0.001; Log-RankTest).
Figure 5.
Pharmacodynamic effects of AZD6244-MK2206 on A549 xenograft mouse models.
The xerograft mouse models were treated with vehicle control, AZD6244 (24 mg/kg), MK2206 (6 mg/kg) or AZD6244-MK2206 (24 mg/kg–6 mg/kg) combination for 3 days. Four hours after the last dose, the tumors were resected, fixed and paraffin-embedded. (A) The sections were analyzed by immunohistochemistry using p-ERK and p-AKT antibodies and the representative photographs in tumor sections are shown. (B) The sections were subjected to TUNEL and DAPI staining. The relative apoptotic cells were determined by counting TUNEL positive cells in five random fields (at 100× magnification) for each sample. Columns, mean; bar, SD. *, P<0.05, compared with the single agent treatments. The representative photographs in tumor sections are shown.