A549 clone sensitivity to combined pemetrexed and methoxyamine treatment before and after IDO induction.

Proliferation of each of 5 individual A549 cell clonal populations before (Panel A) and after (Panel B) IDO induction with IFNγ. A549 clonal populations were cultured with or without IFNγ (25 ng/ml) for 48 h. Cultured medium was then replaced with fresh growth medium containing pemetrexed (30 nM) and methoxyamine (MX) (3 mM). Tumor cells were then allowed to proliferate for 72 h, then enumerated. White bars: A549 clones transfected with scrambled, non-targeting control shRNA. Gray bars: A549 cells transfected with anti-IDO shRNA. Each bar represents the mean of 3 values (n = 3) ± SD. *Significant difference, Student's t-test, p<0.05. Results are normalized to control cells not treated with pemetrexed and methoxyamine, without (panel A) or with (panel B) IFNγ treatment. Panel C: Induction of IDO in A549 clonal cell induces resistance to combined pemetrexed (30 nM) and MX (3 mM) treatment. Results were obtained from 3 independent clonal cell populations with scrambled control shRNA or anti-IDO shRNA, and each bar represents a mean of 9 (white bars) or 6 (black bars) values ± SEM (*p<0.05). Panel D: relationship between IDO protein (relative to actin) and clonal population resistance to combined pemetrexed and MX treatment proliferation relative to untreated control cells). The R2 value of 0.72 represents a moderate positive relationship.