The Complex Dynamic of Phase I Drug Metabolism in the Early Stages of Doxorubicin Resistance in Breast Cancer Cells

Abstract

The altered activity of drug metabolism enzymes (DMEs) is a hallmark of chemotherapy resistance. Cytochrome P450s (CYPs), mainly CYP3A4, and several oxidoreductases are responsible for Phase I metabolism of doxorubicin (DOX), an anthracycline widely used in breast cancer (BC) treatment. This study aimed to investigate the role of Phase I DMEs involved in the first stages of acquisition of DOX-resistance in BC cells. For this purpose, the expression of 92 DME genes and specific CYP-complex enzymes activities were assessed in either sensitive (MCF-7 parental cells; MCF-7/DOX^S) or DOX-resistant (MCF-7/DOX^R) cells. The DMEs genes detected to be significantly differentially expressed in MCF-7/DOX^R cells (12 CYPs and eight oxidoreductases) were indicated previously to be involved in tumor progression and/or chemotherapy response. The analysis of CYP-mediated activities suggests a putative enhanced CYP3A4-dependent metabolism in MCF-7/DOX^R cells. A discrepancy was observed between CYP-enzyme activities and their corresponding levels of mRNA transcripts. This is indicative that the phenotype of DMEs is not linearly correlated with transcription induction responses, confirming the multifactorial complexity of this mechanism. Our results pinpoint the potential role of specific CYPs and oxidoreductases involved in the metabolism of drugs, retinoic and arachidonic acids, in the mechanisms of chemo-resistance to DOX and carcinogenesis of BC.

Keywords: breast cancer (BC); cytochrome P450 (CYP); doxorubicin (DOX); drug metabolism enzymes (DMEs); drug resistance (DR); oxidoreductases.

Figure 1

Differences of mRNA levels of target genes in MCF-7/DOX^R cells. (A) Volcano plots representing the differences in fold change of genes differentially expressed by MCF-7/DOX^R 25nM or 35nM cells, relative to the parental MCF-7/DOX^S cells. Transcripts levels were considered differentially expressed when p < 0.05 and fold change > 2 (upregulated, in green) or <2 (downregulated, in red). POR (CPR gene) and CYP3A4 transcript levels are depicted in blue and purple, respectively. (B) Histograms representing the statistically significant increase (fold change) of target-gene expressions in MCF-7/DOX^R 25 nM or 35 nM relative to the MCF-7/DOX^S cells (technical replicates, N = 2). ALOXE 3: arachidonate lipoxygenase 3; BCMO1: β-carotene 15,15’-monooxygenase 1; CYB5R1: NADH-cytochrome b₅ reductase 1; CYP: cytochrome P450 (isoforms); FDX1: ferredoxin 1; FDXR: ferredoxin reductase; FMO5: flavin containing dimethylaniline monooxygenase 5; KMO: kynurenine 3-hydroxylase; PAH: phenylalanine hydroxylase.

Figure 2

Normalized relative velocities of the CYP activity assays in MCF-7/DOX^R 25 nM microsomal fractions. Values are represented as mean ± SD of technical replicates (N = 3) (* p < 0.05; ** p < 0.005). MROD (red) and EROD (orange) mediated mainly by CYP1A1 (particularly EROD), 1A2 (particularly MROD), and to a minor extent by 1B1; CECOD (green) mediated particularly by CYP1A1, 1A2, and to a minor extent by 2C9, 2C19, and 2D6; C7H (blue) mediated by CYP2A6; DBFOD (purple) mediated mainly by CYP3A4, and to a minor extent by 3A5, 2C8, 2C9, and 2C19. CYP isoenzymes particularly relevant in the specific activities studied are underlined.