Abstract
5-Aminolevulinic acid (ALA) is a natural heme precursor metabolized into protoporphyrin IX (PpIX). PpIX preferentially accumulates in tumor cells resulting in the formation of singlet oxygen upon exposure to visible light. Doxil®, an active agent against breast and ovarian cancer, is a nano-formulation of doxorubicin. This study aimed to investigate in vitro synergistic cytotoxic effect of low doses of combined chemotherapy and ALA/PDT to human breast adenocarcinoma cells (MCF-7) compared to high doses of each individual therapy.
MCF-7 cells were pretreated with Doxil® (48h) followed by ALA/PDT (4h). The cell viability was evaluated by trypan blue assay and PpIX production was measured spectrofluorometrically. Alkaline phosphatase was determined as a marker for cellular differentiation. Apoptosis and necrosis were evaluated by fluorescence stains. The apoptosis cell death pathways were investigated: detection of mitochondrial membrane potential (ΔΨm) and percent of DNA fragmentation, malondialdehyde, histone deacetylase (HDAC) activity, caspase-3 and death receptors (DR4 and DR5). Vascular endothelial growth factor (VEGF) was determined by ELISA, as an angiogenic mediator.
There was a higher reduction in cell viability in Doxil®+ALA/PDT-treated cells compared with their individual effect. The combined therapy showed enhanced apoptosis with a significant increase in the loss of ΔΨm, DNA fragmentation %, caspase-3, DR4, DR5 and lipid peroxides and inhibited HDAC. Pretreatment with Doxil® resulted in a twofold increase in the intracellular PpIX, by increasing the PDT killing of MCF-7 cells.
The combined therapy using 50% of IC50 of ALA/PDT and Doxil® possessed a synergistic apoptotic effect on MCF-7 cells compared to 100% of IC50 of each therapy through enhancing both intrinsic and extrinsic apoptotic pathways, thus may minimize side effects of Doxil® and ALA.