Abstract
5-Aminolaevulinic acid (ALA) and its derivatives act as precursors of the photosensitizer protoporphyrin IX (PpIX). In this study, we compared cytotoxic effects of photodynamic therapy (PDT) with the hexenyl ester of ALA (ALA-hx) between MCF-7 human breast cancer cells and adriamycin-resistant MCF-7 (MCF-7/ADR) cells. Cell viability and apoptosis were determined by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenylte-trazolium bromide (MTT), flow cytometry assays. Chick chorioallantoic membrane (CAM) assays were applied to assess in vivo effect of ALA-hx PDT. Molecular analyses using Western blots and minimal reporter constructs containing the antioxidant response element (ARE) region were performed to reveal mechanistic basis for the differential PDT sensitivity of MCF-7 and MCF-7/ADR cells. In MCF-7/ADR cells, PDT with ALA-hx more efficiently produced reactive oxygen species (ROS) and suppressed cell viability compared to MCF-7 cells. Cell death induced by ALA-hx PDT in MCF-7/ADR cells was mainly due to apoptosis. CAM assays confirmed that the apoptotic activity of PDT in MCF-7/ADR cells was significantly higher than that in control MCF-7 cells. We also found that MCF-7/ADR cells produced lower levels of glutathione (GSH), a major antioxidant, than control MCF-7 cells. Expression of Nrf2-dependent anti-oxidant genes including γ-glutamylcysteine ligase, heme oxygenase-1, and quinone oxidoreductase were down-regulated in MCF-7/ADR cells, and Nrf2 overexpression partially decreased the susceptibility of ALA-hx PDT in MCF-7/ADR cells. Moreover, PpIX synthesis and expression levels of protoporphyrinogen oxidase (PPO) and coproporphyrinogen oxidase (CPO) were much higher in MCF-7/ADR cells than MCF-7 cells. ALA-hx PDT more potently produced intracellular ROS in MCF-7/ADR cells, which might be due to down-regulation of Nrf2-mediated anti-oxidant gene transcription and up-regulation of PpIX synthesis via the induction of CPO and PPO. These findings suggest that ALA-hx PDT may be usable as a therapeutic alternative for adriamycin-resistant breast cancer.
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