Subcellular Quantification Of Doxorubicin And Its Metabolite Using Liquid Chromatography-Tandem Mass Spectrometry

Doxorubicin (DOX) is widely used in the world as an anticancer agent for the treatment of leukemia and solid tumors. However, its clinical use is largely limited by the emergence of cardiotoxicity. One of the most frequently proposed mechanisms for DOX induced cardiotoxicity is the formation of metabolites. However, the enzymatic pathways involved in DOX intracellular metabolism have not been fully elucidated thus far. To provide a more detailed description of DOX metabolism, an assay using liquid chromatography-tandem mass spectrometry (LC/MS/MS) was developed in our lab to simultaneously determine DOX and its primary metabolite doxorubicinol (DOXol) in subcellular compartments.Sample cleanup and enrichment were achieved using solid phase extraction, and the validated calibration ranges for DOX and DOXol were 5.00-1000 ng/mL and 0.50-50.0 ng/mL, respectively. Good accuracy and precision were achieved. Using this assay, the accumulation of DOX and DOXol in whole cells, nuclear-enriched fraction (NEF) and organelle-enriched fraction (OEF) were compared between two human T leukemia cell lines (i.e., Jurkat and CCRF-CEM). A time-course analysis was also carried out. The resulting varieties of DOX and DOXol subcellular distributions and concentration-time profiles might be attributed to the differential expression, activities, and localization of reductive enzymes within these cell lines.Most current separation techniques are not applicable to simultaneously and efficiently prepare organelles with high purity and yield. Therefore, an immunoisolation assay was developed here. Using indirect coating, cell lysates were labeled with primary antibody anti-voltage-dependent anion-selective channel 1(VDAC1) first and subsequently incubated with magnetic beads coated with secondary antibody. After a separation in a high gradient magnetic field, the organelles were obtained with high purity, yield and integrity. To be specific, there is no evidence of cross contamination between fractions. The maximum mitochondria recovered were approximately 65%. Cytochrome C and cathepsin D, as indicators of membrane permeabilization of mitochondria and acidic organelles, were not observed in the cytoplasmic fractions. Using this immunoisolation assay together with LC/MS/MS, the subcellular distribution profiles of DOX and DOXol in mitochondria and acidic organelles of human breast cancer cells MCF-7/WT and MCF-7/ADR were determined. In addition, these distributions were further evaluated using verapamil and NH4Cl.To illustrate the enzymatic pathways involved in DOX metabolism, we established overexpressed MCF-7 cells (i.e., Aldo-keto reductase 1C3, AKR1C3 transfected). Using the developed LC/MS/MS assay, the conversion of DOX to its metabolite DOXol in MCF-7/AKR1C3 was compared with that in MCF-7 wide type cells. It was found that the ratio of the DOXol to DOX was elevated, which was highly significant, with P value of 0.0373. Such changes revealed that AKR1C3 may play an important role in the DOX transformation.