The Adaptation of MCF-7 Breast Cancer Spheroids to the Chemotherapeutic Doxorubicin: The Dynamic Role of Phase I Drug Metabolizing Enzymes

<b>Background/Objectives:</b> Drug resistance (DR) is a major challenge in cancer therapy, contributing to approximately 90% of cancer-related deaths. While alterations in drug metabolism are known to be key drivers of DR, their role—particularly in the early stages of acquired chemoresistance—remains understudied. Phase I drug-metabolizing enzymes (DMEs), especially cytochrome P450s (CYPs), significantly influence the metabolic fate of chemotherapeutic agents, directly affecting drug response. This study aimed to investigate the role of Phase I DMEs in the early metabolic adaptation of breast cancer (BC) MCF-7 cells to doxorubicin (DOX). <b>Methods:</b> Four types of spheroids were generated from MCF-7 cells that were either DOX-sensitive (DOX^S) or adapted to low concentrations of the chemotherapeutic agent (DOX^A 25, 35, and 45 nM). The expression levels of 92 Phase I DMEs and the activities of specific CYP isoforms were assessed in both DOX^S and DOX^A spheroids. <b>Results:</b> A total of twenty-four DMEs, including fifteen CYPs and nine oxidoreductases, were found to be differentially expressed in DOX^A spheroids. Pathway analysis identified key roles for the differentially expressed DMEs in physiologically relevant pathways, including the metabolism of drugs, arachidonic acid, retinoic acid, and vitamin D. <b>Conclusions:</b> The deconvolution of these pathways highlights a highly dynamic process driving early-stage DOX resistance, with a prominent role of CYP3A-dependent metabolism in DOX adaptation. Our findings provide valuable insights into the underlying molecular mechanisms driving the early adaptation of MCF-7 cells to DOX exposure.