title: Synergistic apoptotic effect of Doxil ® and aminolevulinic acid-based photodynamic therapy on human breast adenocarcinoma cells.
abstract: BACKGROUND
  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.
  
  
  METHODS
  MCF-7 cells were pretreated with Doxil(®) (48 h) 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.
  
  
  RESULTS
  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.
  
  
  CONCLUSIONS
  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.
authors.affiliation: Department of Pharmacology and Toxicology, Faculty of Pharmacy, October 6 University, October 6 City, Giza, Egypt.
authors.forename: Soad
authors.initials: S
authors.lastname: Zakaria
authors.affiliation: Cancer Biology Laboratory, Center of Excellence for Advanced Sciences, National Research Center, Cairo, Egypt; Department of Biochemistry, National Research Center, Cairo, Egypt. Electronic address: aeldeen7@yahoo.com.
authors.forename: Amira M
authors.initials: AM
authors.lastname: Gamal-Eldeen
authors.affiliation: Cancer Biology Laboratory, Center of Excellence for Advanced Sciences, National Research Center, Cairo, Egypt; Department of Medical Biochemistry, National Research Center, Cairo, Egypt.
authors.forename: Sherien M
authors.initials: SM
authors.lastname: El-Daly
authors.affiliation: Department of Pharmacology and Toxicology, Faculty of Pharmacy, Cairo University, Cairo, Egypt.
authors.forename: Samira
authors.initials: S
authors.lastname: Saleh
mesh_terms: D000230:Adenocarcinoma / Q000188:drug therapy* / Q000378:metabolism* / Q000473:pathology; D000622:Aminolevulinic Acid / Q000008:administration & dosage*; D000903:Antibiotics, Antineoplastic / Q000008:administration & dosage; D017209:Apoptosis / Q000187:drug effects; D001943:Breast Neoplasms / Q000188:drug therapy* / Q000378:metabolism* / Q000473:pathology; D002470:Cell Survival / Q000187:drug effects; D004305:Dose-Response Relationship, Drug; D004317:Doxorubicin / Q000008:administration & dosage / Q000031:analogs & derivatives*; D004357:Drug Synergism; D004359:Drug Therapy, Combination / Q000379:methods; D006801:Humans; D061986:MCF-7 Cells; D011092:Polyethylene Glycols / Q000008:administration & dosage; D011838:Radiation-Sensitizing Agents / Q000008:administration & dosage; D053218:Receptors, Death Domain / Q000378:metabolism*; D016896:Treatment Outcome
chemical_list: D000903:Antibiotics, Antineoplastic; D011838:Radiation-Sensitizing Agents; D053218:Receptors, Death Domain; C506643:liposomal doxorubicin; D011092:Polyethylene Glycols; D004317:Doxorubicin; D000622:Aminolevulinic Acid
keywords: 5-Aminolevulinic acid; Apoptosis; Breast cancer; Death receptors; Doxil(®); Protoporphyrin IX