In vitro study on the schedule-dependency of the interaction between pemetrexed, gemcitabine and irradiation in non-small cell lung cancer and head and neck cancer cells

Abstract

Background: Based on their different mechanisms of action, non-overlapping side effects and radiosensitising potential, combining the antimetabolites pemetrexed (multitargeted antifolate, MTA) and gemcitabine (2',2'-difluorodeoxycytidine, dFdC) with irradiation (RT) seems promising. This in vitro study, for the first time, presents the triple combination of MTA, dFdC and irradiation using various treatment schedules.

Methods: The cytotoxicity, radiosensitising potential and cell cycle effect of MTA were investigated in A549 (NSCLC) and CAL-27 (SCCHN) cells. Using simultaneous or sequential exposure schedules, the cytotoxicity and radiosensitising effect of 24 h MTA combined with 1 h or 24 h dFdC were analysed.

Results: Including a time interval between MTA exposure and irradiation seemed favourable to MTA immediately preceding or following radiotherapy. MTA induced a significant S phase accumulation that persisted for more than 8 h after drug removal. Among different MTA/dFdC combinations tested, the highest synergistic interaction was produced by 24 h MTA followed by 1 h dFdC. Combined with irradiation, this schedule showed a clear radiosensitising effect.

Conclusions: Results from our in vitro model suggest that the sequence 24 h MTA --> 1 h dFdC --> RT is the most rational design and would, after confirmation in an in vivo setting, possibly provide the greatest benefit in the clinic.

Figure 1

MTA combined with irradiation. (A) Combination index (CI) plots of radiation (RT) immediately followed by 24 h incubation with various concentrations of MTA in A549 cells. The plots represent the mean of at least three independent experiments. (B-C) Radiation dose-response curves of CAL-27 (B) and A549 (C) cells treated with 24 h MTA followed by different time intervals (24, 8, 4, 1, 0 h) and subsequent irradiation. Survival values from combination experiments were normalised to MTA cytotoxicity (see table 1 for the cytotoxic effect of MTA alone). Data points represent mean values from at least three independent experiments; bars, SD.

Figure 2

Influence of MTA on cell cycle distribution. DNA histograms of A549 cells at different time points after 24 h incubation with 100 nM MTA.

Figure 3

Combination index (CI) plots of MTA - dFdC combinations in CAL-27 (A-C) and A549 (D-F) cells. Cells were treated with a concentration range of MTA for 24 h (0-100 nM for CAL-27 cells; 0-2000 nM for A549 cells) combined with a constant concentration of dFdC for 24 h (2 or 4 nM) (A, D). Alternatively, cells were incubated with a concentration range of dFdC (0-100 nM for 24 h (B, E) or 0-5 μM for 1 h (C, F)) combined with a constant concentration of MTA (50 or 100 nM for CAL-27; 200 or 500 nM for A549). The plots represent the mean of at least three independent experiments.

Figure 4

Inhibitory effect of 24 h MTA - 1 h dFdC combinations on cell survival of CAL-27 (A, B) and A549 (C, D) tumour cells. Survival curves were corrected for the cytotoxic effect of 24 h MTA alone. In CAL-27, 24 h incubation with 50 or 100 nM MTA resulted in resp. 90 ± 7% and 73 ± 8% survival. In A549, 24 h incubation with 200 or 500 nM MTA resulted in resp. 73 ± 10% and 50 ± 7% survival. Data points represent mean values from at least three independent experiments; bars, SD.