Contribution of Autophagy in Acquired Drug Resistance of Human Breast Cancer Cells MCF7 to Doxorubicin

Abstract

Introduction:

At present, autophagy has attracted increased attention as a potential therapeutic target for breast cancer. Many investigators are trying to overcome drug resistance by inhibition of autophagy. The development of in vitro drug-resistant cell lines with well-characterized autophagy-related markers is required to discover new autophagy inhibitors. Therefore, in this study we established resistant human breast cancer cells MCF7 to doxorubicin (DOX) and used flow cytometric assays to study autophagic flux.

Materials and Methods:

Resistant MCF7 subline (MCF7.res) was derived from parental MCF7 cells (MCF7.par) by continuous exposure to stepwise increasing concentrations of DOX (30–54 μM). Autophagic flux was assessed by the antibody labeling of the microtubule-associated protein LC3-II and analyzed by flow cytometry. In addition, lysosomal mass was measured flow cytometrically by LysoTracker Green (LTG) staining. The median fluorescence intensity of anti-LC3-II and LTG was compared between parental and resistant MCF7 cells. For detection of apoptotic cell death, Annexin V/propidium iodide staining was performed. After ∼6 months, MCF7.res subline was obtained from MCF7.par cells.

Results:

DOX resistance was confirmed by measurement of fold resistance and growth curve analysis. Our established MCF7.res subline exhibited 7.1-fold resistance to DOX compared with MCF7.par cells. Flow cytometric analysis revealed that LC3-II level and LTG signal were elevated in MCF7.res compared with MCF7.par, elucidating increased autophagic flux. The results of apoptosis assay showed that chloroquine (an autophagy inhibitor) could reverse drug resistance of MCF7.res by inhibiting autophagy.

Conclusion:

Our established DOX-resistant MCF7 cells model is reliable and applicable for investigating new autophagy inhibitors.