Molecular mechanisms underlying the interaction between ZD1839 ('Iressa') and cisplatin/5-fluorouracil

Abstract

ZD1839 ('Iressa'), an orally active, selective epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor, is currently being investigated in clinical trials as a treatment for cancer. 'Iressa' is a trademark of the AstraZeneca group of companies. We have previously demonstrated a synergistic interaction between ZD1839 and cisplatin/5-fluorouracil (5FU) in CAL33, a human head and neck cancer cell line that markedly expresses EGFR. This study examined the effects of this drug combination on the cell cycle, cell cycle regulators, apoptosis-related factors, EGFR-related signalling and DNA repair in CAL33 cells. The cells were incubated with ZD1839 alone for 48 h, then cisplatin and 5FU were added. Exposure to the drug combination continued for a further 48 h. ZD1839 alone induced accumulation of cells in the G0/G1 phase of the cell cycle at 24 h accompanied by a concomitant increase in p21, p27 and Bax, a significant decrease in Bcl2 and a decrease in Akt phosphorylation. A decrease in DNA-PK was observed at 48 h. ZD1839 alone had no effect on caspase-3 activity, but addition of ZD1839 to cisplatin-5FU led to a significant increase in caspase-3 activity at 96 h. Thus, ZD1839 affects key cellular pathways controlling cell proliferation, apoptosis and DNA repair. These data provide a rationale to support clinical trials combining ZD1839 and cisplatin-5FU and other protocols that combine EGFR-targeting agents with chemotherapy or radiotherapy.

Figure 1

Study design. Single-drug exposures: 48 h after seeding, cells were exposed to ZD1839 alone for 96 h (condition ZD1839); 48 h after seeding, cells were exposed to medium alone for 48 h, followed by cisplatin–5FU for a further 48 h (condition CPFU). Drug combination: 48 h after seeding, CAL33 cells were exposed to ZD1839 (ZD) alone at its IC₅₀ (6 μM) for 48 h, then exposed to cisplatin and 5FU at their IC₃₀'s in combination with ZD1839 for a further 48 h (condition ZDCPFU).

Figure 9

Representative Western blots related to Figures 2, 3, 4 and 8. For details on these analyses, see Materials and Methods section.

Figure 2

Impact of ZD1839 (6 μM) and cisplatin–5FU (0.15 and 3.3 μM, respectively) on cell proliferation and related molecular markers: (A) time-related impact of drugs on cell proliferation, (B) effect of ZD1839 on cell cycle at 24 h, (C) influence of drugs and drug combinations on p21 and (D) influence of drugs and drug combinations on p27. White bars=controls; black bars=ZD1839; dark grey bars=cisplatin–5FU combination; clear grey bars=ZD1839–cisplatin–5FU combination. Combination schedule as indicated in the legend to Figure 1.

Figure 3

Impact of ZD1839 on key pathways regulated by EGFR signalling. (A) ZD1839 (6 μM, IC₅₀ value) diminished MAP kinase phosphorylation. (B) ZD1839 (6 μM, IC₅₀ value) diminished AKT phosphorylation. ^*P<0.05 vs control, bars indicate standard deviation from the mean of three separate experiments.

Figure 4

ZD1839 and cisplatin–5FU alone caused an upregulation of Bax (A) and a downregulation of Bcl2 (B), and no additive effect was observed when ZD1839 was combined with cisplatin–5FU. Results confirmed by the Bax/Bcl2 ratio (C). ^*P<0.05 vs control, bars indicate standard deviation from the mean of three separate experiments.

Figure 5

Impact of the applied drugs on mitochondrial energisation.

Figure 6

Impact of drugs and drug combination on caspase 3 activity. ZD1839 alone had no effect on caspase 3 activity, but the combination of ZD1839 with cisplatin–5FU led to a supra-additive increase in caspase 3 activity at 96 h. ^*P<0.05 vs CFPU combination, bars indicate standard deviation from the mean of three separate experiments.

Figure 7

Impact of drugs and drug combination on p53. ^*P<0.05 vs control, bars indicate standard deviation from the mean of three separate experiments.

Figure 8

Impact of drugs and drug combination on DNA-PK. ^*P<0.05 vs control, bars indicate standard deviation from the mean of three separate experiments.