Methotrexate modulates folate phenotype and inflammatory profile in EA.hy 926 cells

Abstract

EA.hy 926 cells grown under low folate conditions adopt a "pro-atherosclerotic" morphology and biochemical phenotype. Pharmacologically relevant doses of the antifolate drug methotrexate (MTX) were applied to EA.hy 926 cells maintained in normal (Hi) and low (Lo) folate culture media. Under both folate conditions, MTX caused inhibition of cell proliferation without significantly compromising metabolic activity. MTX treated Hi cells were depleted of folate derivatives, which were present in altered proportions relative to untreated cells. Transcript profiling using microarrays indicated that MTX treatment modified the transciptome in similar ways for both Hi and Lo cells. Many inflammation-related genes, most prominently those encoding C3 and IL-8, were up-regulated, whereas many genes involved in cell division were down-regulated. The results for C3 and IL-8 were confirmed by quantitative RT-PCR and ELISA. MTX appears to modify the inflammatory potential of EA.hy 926 cells such that its therapeutic properties may, at least under some conditions, be accompanied by the induction of a subset of gene products that promote and/or maintain comorbid pathologies.

Keywords: Folate; Gene expression; Immune modulation; Inflammation; Methotrexate.

Figure 1

Folate/Homocysteine pathway. 5-MTHF, 5-methyltetrahydrofolate; 5,10-MTHF, 5,10-methenyltetrahydrofolate; DHF, dihydrofolate; DHFR, dihydrofolate reductase; dTMP, deoxythymidine monophosphate; dUMP, deoxyuridine monophosphate; Hcy, homocysteine; MTX, methotrexate; SAH, S-adenosylhomocysteine; SAM, S-adenosylmethionine; THF, tetrahydrofolate; TYMS, thymidylate synthase.

Figure 2

The effects of MTX dose on the proliferation and metabolic activity of EA.hy 926 cells maintained in Hi and Lo folate media. A and B, Newly plated EA.hy 926 Hi (A) and Lo (B) cells were incubated overnight and plating medium was replaced with Hi or Lo medium, respectively, containing 0, 0.1, 0.25, or 0.5 μM MTX. After 24 and 48 h the medium was removed and adherent cells were fixed and stained according to the manufacturer’s instructions for BrdU quantitation. Each bar represents the mean ± S.D. of three samples. This experiment is representative of a total of three experiments with similar results. C and D, Confluent Hi (C) and Lo (D) cells were incubated with 0, 0.1, 0.25, or 0.5 μM MTX. Cells were incubated with alamar blue for the 2 h immediately prior to the reading of absorbance at the following time points: 2, 8, 16, 24, and 48 h of exposure to MTX. Absorbance was measured at 570nm with a correction at 630nm and calculated as the percentage of control. Each bar represents the mean ± S.D. of three samples. This experiment is representative of a total of three experiments with similar results. *P values <0.05 compared with control at the same time point.

Figure 2

The effects of MTX dose on the proliferation and metabolic activity of EA.hy 926 cells maintained in Hi and Lo folate media. A and B, Newly plated EA.hy 926 Hi (A) and Lo (B) cells were incubated overnight and plating medium was replaced with Hi or Lo medium, respectively, containing 0, 0.1, 0.25, or 0.5 μM MTX. After 24 and 48 h the medium was removed and adherent cells were fixed and stained according to the manufacturer’s instructions for BrdU quantitation. Each bar represents the mean ± S.D. of three samples. This experiment is representative of a total of three experiments with similar results. C and D, Confluent Hi (C) and Lo (D) cells were incubated with 0, 0.1, 0.25, or 0.5 μM MTX. Cells were incubated with alamar blue for the 2 h immediately prior to the reading of absorbance at the following time points: 2, 8, 16, 24, and 48 h of exposure to MTX. Absorbance was measured at 570nm with a correction at 630nm and calculated as the percentage of control. Each bar represents the mean ± S.D. of three samples. This experiment is representative of a total of three experiments with similar results. *P values <0.05 compared with control at the same time point.

Figure 3

The effect of MTX on FA and the folate derivatives: 5-MTHF, THF, and 5,10-MTHF. Folate derivatives were measured by LC/MRM/MS in lysates from confluent Hi (A) and Lo (B) cells following treatment with 0.5 μM MTX for 48 h. Each bar represents the mean ± S.D. of three samples. This experiment is representative of a total of three experiments with similar results except that in the other two experiments MTX treated Lo cells had slightly decreased levels of folate derivatives relative to control cells. *P values <0.05 compared with control.

Figure 4

C3 and IL-8 mRNA levels in Hi and Lo cells treated with 0.5 μM MTX for 48 h. C3 and IL-8 mRNA levels were assessed by qRT-PCR using the mRNA levels of the endogenous housekeeping gene GAPDH for normalization. Each bar represents mean ± S.D. target mRNA expression levels normalized to GAPDH mRNA levels of three samples. This experiment is representative of a total of three experiments with similar results; C3 mRNA (A), IL-8 mRNA (B). *P values <0.05 compared with respective control. #P values <0.05 for untreated Hi cells compared to untreated Lo cells.

Figure 5

Secreted C3, IL-8, and MCP-1 protein levels in Hi and Lo cell media. Hi and Lo cells were treated with 0.5 μM MTX for 48 h. C3 (A), IL-8 (B) and MCP-1 (C) were measured by ELISA. Each bar represents the mean ± S.D. of three samples. These data are representative of a total of three experiments with similar results. *P values <0.05 compared with respective control. #P values <0.05 for untreated Hi cells compared to untreated Lo cells.