Pemetrexed alters folate phenotype and inflammatory profile in EA.hy 926 cells grown under low-folate conditions

Abstract

Elevated homocysteine is a risk marker for several major human pathologies. Emerging evidence suggests that perturbations of folate/homocysteine metabolism can directly modify production of inflammatory mediators. Pemetrexed acts by inhibiting thymidylate synthetase (TYMS), dihydrofolate reductase (DHFR), and glycinamide ribonucleotide formyltransferase (GARFT). EA.hy 926 cells grown under low ("Lo") and high ("Hi") folate conditions were treated with pemetrexed. The concentrations of several intracellular folate derivatives were measured using LC-MRM/MS. Lo cells had lower total folate concentrations and a different distribution of the intracellular folate derivatives than Hi cells. Treatment with pemetrexed caused a decrease in individual folate analytes. Microarray analysis showed that several genes were significantly up or down-regulated in pemetrexed treated Lo cells. Several of the significantly up-regulated transcripts were inflammatory. Changes in transcript levels of selected targets, including C3, IL-8, and DHFR, were confirmed by quantitative RT-PCR. C3 and IL-8 transcript levels were increased in pemetrexed-treated Lo cells relative to Lo controls; DHFR transcript levels were decreased. In Lo cells, IL-8 and C3 protein concentrations were increased following pemetrexed treatment. Pemetrexed drug treatment was shown in this study to have effects that lead to an increase in pro-inflammatory mediators in Lo cells. No such changes were observed in Hi cells, suggesting that pemetrexed could not modify the inflammatory profile in the context of cellular folate sufficiency.

Fig. 1

Folate/homocysteine metabolic pathway. 5-MTHF, 5-methyltetrahydrofolate; THF, tetrahydrofolate; 5,10-MTHF, 5,10-methenyltetrahydrofolate; 5,10-MeTHF, 5,10-methylenetetrahydrofolate; FA, folic acid; DHF, dihydrofolate; Hcy, homocysteine; dUMP, deoxyuridine monophosphate; dTMP, deoxythymidine monophosphate; SAH, S-adenosylhomocysteine; SAM, S-adenosylmethionine; DHFR, dihydrofolate reductase; TYMS, thymidylate synthase; PMX, pemetrexed.

Fig. 2

LC-MRM/MS measurement of folate derivatives. Lo and Hi cells were exposed to 0.5 μM pemetrexed for 48 h. Results represent the mean±S.D. of biological triplicates. (A) Hi cells, (B) Lo cells. *P values <0.05 compared with control. PMX, pemetrexed.

Fig. 3

Assessment of DHFR, C3, MCP-1 and IL-8 transcripts in Lo and Hi cells treated with pemetrexed by qRT-PCR. Lo and Hi cells were exposed to 0.5 μM pemetrexed for 48 h. Expression of GAPDH was used for normalization. Results represent the mean±S.D. of biological triplicates. (A) DHFR, (B) C3, (C) MCP-1, (D) IL-8. *P values <0.05 compared with respective control. ^#P values <0.05 for Hi control compared to Lo control. PMX, pemetrexed.

Fig. 4

Assessment of C3, MCP-1 and IL-8 protein in media of Lo and Hi cells treated with pemetrexed by ELISA. Lo and Hi cells were exposed to 0.5 μM pemetrexed for 48 h. Secreted proteins were measured in media. Results represent the mean±S.D. of biological triplicates. (A) C3, (B) IL-8, (C) MCP-1. *P values <0.05 compared with respective control. ^#P values <0.05 for Hi control compared to Lo control. PMX, pemetrexed.