Effect of tumour necrosis factor-alpha on estrogen metabolic pathways in breast cancer cells

Abstract

Tumor necrosis factor-alpha (TNF-α) is a proinflammatory cytokine that has been linked to breast cancer development. Estrogen metabolic pathway is also involved in breast carcinogenesis and DNA adducts formation. In this study we investigated the effect of TNF-α on the estrogen metabolic pathway in MCF-7, a breast cancer cell line. Capillary liquid chromatography/mass spectrometry (LC/MS) and High performance liquid chromatography (HPLC) were used for analysis of estrogen metabolites and estrogen-DNA adducts levels respectively. Reporter gene assay, Real time reverse transcription polymerase chain reaction (real time RT-PCR) and Western blot were used to assess the expression of estrogen metabolizing genes and enzymes. TNF-α significantly increased the total EM and decreased the estrone (E1) / 17-β estradiol (E2) ratio. Moreover, it altered the expression of genes and enzymes involved in E2 activation and deactivation pathways e.g. Cytochrome P-450 1A1 (CYP1A1), Cytochrome P-450 1B1 (CYP1B1), Catechol-O-methyl transferase (COMT) and Nicotinamide adenine dinucleotide phosphate-quinone oxidoreductase 1 (NQO1). In addition, there were increased levels of some catechol estrogens e.g. 4-hydroxy-estrone (4-OHE1) and 2-hydroxyestradiol (2-OHE2) with decreased levels of methylated catechols e.g. 2-methoxy estradiol (2-MeOE2). DNA adducts especially 4-OHE1-[2]-1-N3 Adenine was significantly increased. TNF-α directs the estrogen metabolism into more hormonally active and carcinogenic products in MCF-7. This may implicate a new possible explanation for inflammation associated breast cancer.

Keywords: Breast cancer; DNA adducts.; Estrogen metabolites; Estrogen metabolizing genes and enzymes; Tumor necrosis factor-alpha.

Figure S1

Part of the metabolic pathway of estradiol and the role of various enzymes involved: Estradiol is metabolized into 2-hydroxyestradiol (2-OHE2) and 4-hdroxyestradiol (4-OHE2) by CYP1A1 and CYP1B1 respectively. These catechols undergo further oxidation into semiquinones and quinones that react with DNA to form depurinating adducts leading to mutations associated with breast cancer. NQO1 reduces these quinones back to catechols which are detoxified into methoxy derivatives by the action of COMT. This protects the cells against DNA adducts formation and lowers the potential for mutagenic damage.

Fig 1

Effect of TNF-α on the ratio of E1/E2 in MCF-7 cells. Cells were grown in serum-free media and treated with E2 (10 nM) alone or with TNF-α (5 ng/ml) for 48 hours. Media were collected to quantify the mean rate of EM formation in (pmole/mg protein/48 hours) by LC/MS. Data were normalized against protein concentration. E1/E2 ratio was calculated. Values represent the Mean±SEM of three experiments. * indicates significant difference of TNF-α treated cells compared to control at P < 0.05.

Fig 2

Effect of TNF-α on depurinating DNA adducts formation. Cells were grown in serum-free media and treated with E2 (10 nM) alone or with TNF-α (5 ng/ml) for 48 hours. The media were collected for DNA adducts analysis by HPLC. Values represent the Mean±SEM of three experiments. * indicates significant difference of TNF-α treated cells compared to control at P < 0.05.

Fig 3

Effect of TNF-α on CYP1A1 in MCF-7 cells. (a) Luciferase reporter gene assay. The cells were stably transfected with reporter plasmids containing CYP1A1 promoters. After treatment with different doses of TNF-α, luciferase production was measured 48 hours later. Results represent the Mean±SEM of two experiments. (b) Real time RT-PCR analysis.The cells were treated with TNF-α 5 ng/ml and 10 ng/ml for 24 hours followed by total RNA isolation from the cells and the CYP1A1 mRNA expression was determined by real time RT-PCR. The threshold cycle value of CYP1A1 was normalized based on that of GAPDH. Results represent the Mean±SEM of two experiments. * indicates significant difference of TNF-α treated cells compared to control at P < 0.05.

Fig 4

Effect of TNF-α on CYP1B1 in MCF-7 cells: (a) Luciferase reporter gene assay. The cells were stably transfected with reporter plasmids containing CYP1B1 promoters. After treatment with different doses of TNF-α, luciferase production was analyzed 48 hours later. Results represent the Mean±SEM of two experiments. (b) Real time RT-PCR analysis. The cells were treated with TNF-α 10 ng/ml for 24 hours followed by total RNA isolation from the cells, and the CYP1B1 mRNA expression was determined by real time RT-PCR. The threshold cycle value of CYP1B1 was normalized based on that of GAPDH. Results represent the Mean±SEM of two experiments. (c) Western blot analysis. CYP1B1 protein levels were determined 72 hours after treatment with different doses of TNF-α. * indicates significant difference of TNF-α treated cells compared to control at P < 0.05.

Fig 5

Effect of TNF-α on COMT in MCF-7 cells: (a) Luciferase reporter gene assay. The cells were stably transfected with reporter plasmids containing COMTP1 (i) and COMTP2 promoters (ii). After treatment with different doses of TNF-α, luciferase production was analyzed 48 hours later. Results represent the Mean±SEM of two experiments. (b) Real time RT-PCR analysis. The cells were treated with TNF-α 10 ng/ml for 24 hours followed by total RNA isolation from the cells and COMT mRNA expression was determined by real time RT-PCR. The threshold cycle value of COMT was normalized based on that of GAPDH. Results represent the Mean±SEM of two experiments. (c) Western blot analysis. Both S-COMT and MB-COMT protein levels were determined 72 hours after treatment with different doses of TNF-α. * indicates significant difference of TNF-α treated cells compared to control at P < 0.05.

Fig 6

Effect of TNF-α on NQO1 in MCF-7 cells: (a) Luciferase reporter gene assay. The cells were stably transfected with reporter plasmids containing NQO1 promoters. After treatment with different doses of TNF-α, luciferase production was measured 48 hours later. Results represent the Mean±SEM of duplicate experiments. (b) Real time RT-PCR analysis. The cells were treated with TNF-α 10 ng/ml for 24 hours followed by total RNA isolation from the cells, and the NQO1 mRNA expression was determined by real time RT-PCR. The threshold cycle value of NQO1 was normalized based on that of GAPDH. Results represent the Mean±SEM of two experiments. (c) Western blot analysis. NQO1 protein levels were determined 72 hours after treatment with different doses of TNF-α. * indicates significant difference of TNF-α treated cells compared to control at P < 0.05.