Abstract
BackgroundThe roles and mechanisms involved in starvation-induced autophagy in mammalian cells have been extensively studied. However, less is known about the potential role for autophagy as a survival pathway in acquired drug resistance in cancer cells under nutrient-rich conditions.MethodsWe selected MCF-7 breast tumor cells for survival in increasing concentrations of doxorubicin and assessed whether the acquisition of doxorubicin resistance was accompanied by changes in doxorubicin and lysosome localization and the activation of autophagy, as assessed by laser scanning confocal microscopy with or without immunohistochemical approaches. The ultrastructure of cells was also viewed using transmission electron microscopy. Cellular levels of autophagy and apoptosis-related proteins were assessed by immunoblotting techniques, while protein turnover was quantified using a flux assay.ResultsAs cells acquired resistance to doxorubicin, the subcellular location of the drug moved from the nucleus to the perinuclear region. The location of lysosomes and autophagosomes also changed from being equally distributed throughout the cytoplasm to co-localizing with doxorubicin in the perinuclear region. There was an apparent temporal correlation between the acquisition of doxorubicin resistance and autophagy induction, as measured by increases in monodansylcadaverine staining, LC3-II production, and co-localization of LAMP1 and LC3-II immunofluorescence. Electron microscopy revealed an increase in cytoplasmic vacuoles containing mitochondria and other cellular organelles, also suggestive of autophagy. Consistent with this view, a known autophagy inhibitor (chloroquine) was highly effective in restoring doxorubicin sensitivity in doxorubicin-resistant cells. Moreover, this induction of autophagy correlated temporally with increased expression of the selective cargo receptor p62, which facilitates the delivery of doxorubicin-damaged mitochondria and other organelles to autophagosomes. Finally, we suggest that autophagy associated with doxorubicin resistance may be distinct from classical starvation-induced autophagy, since Beclin 1 and Atg7 expression did not change upon acquisition of doxorubicin resistance, nor did recombinant Bcl2 overexpression or an Atg7 knockdown alter doxorubicin cytotoxicity.ConclusionTaken together, our findings suggest that doxorubicin resistance in MCF-7 breast cancer cells is mediated, at least in part, by the activation of autophagy, which may be distinct from starvation-induced autophagy.
Highlights
The roles and mechanisms involved in starvation-induced autophagy in mammalian cells have been extensively studied
Acquisition of resistance to doxorubicin in MCF-7 cells and restoration of sensitivity by chloroquine treatment Using clonogenic assays to measure drug sensitivity, we observed that resistance to doxorubicin in MCF-7 cells was acquired when the doxorubicin selection concentration reached or exceeded a specific ‘threshold’ dose, as we described previously [35]
Our findings suggest that upon acquisition of doxorubicin resistance, autophagy is increased through changes in cellular Microtubule-associated protein light chain 3 (LC3)-II levels and localization, as exhibited by the change from a weak, diffuse, punctate pattern to strong clustered staining in the perinuclear region
Summary
The roles and mechanisms involved in starvation-induced autophagy in mammalian cells have been extensively studied. Less is known about the potential role for autophagy as a survival pathway in acquired drug resistance in cancer cells under nutrient-rich conditions. A basal level of autophagy occurs in all cells to ensure that only functional organelles are retained [4]. Autophagy permits cells to survive by metabolizing their own organelles as a source of energy. This survival mechanism is considered a “double-edged sword”, since cells can die by prolonged autophagy ( referred to as type II programmed cell death) [14,15,16]
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