title: Development of rapid and highly sensitive HSPA1A promoter-driven luciferase reporter system for assessing oxidative stress associated with low-dose photodynamic therapy.
abstract: Photodynamic therapy (PDT) is a regulatory-approved modality for treating a variety of malignant tumors. It induces tumor tissue damage via photosensitizer-mediated oxidative cytotoxicity. The heat shock protein 70 (HSP70-1) is a stress protein encoded by the HSPA1A gene and is significantly induced by oxidative stress associated with PDT. The aim of this study was to identify the functional region of the HSPA1A promoter that responds to PDT-induced oxidative stress and uses the stress responsiveness of HSPA1A expression to establish a rapid and cost-effective photocytotoxic assessment bioassay to evaluate the photodynamic potential of photosensitizers. By constructing luciferase vectors with a variety of hspa1a promoter fractions and examining their relative luciferase activity, we demonstrated that the DNA sequence from -218 to +87 of the HSPA1A gene could be used as a functional promoter to detect the PDT-induced oxidative stress. The maximal relative luciferase activity level of HSPA1A (HSP70-1) induced by hypericin-PDT was nearly nine times that of the control. Our results suggest that the novel reporter gene assay using a functional region of the HSP70A1A promoter has significant advantages for the detection of photoactivity in terms of both speed and sensitivity, when compared with a cell viability test based on ATP quantification and ROS levels. Furthermore, phthalocyanine zinc and methylene blue both induced significantly elevated levels of relative luciferase activity in a dose-dependent manner.
authors.affiliation: State Key Laboratory of Bioreactor Engineering and Shanghai Key Laboratory of Chemical Biology, School of Pharmacy, East China University of Science and Technology, 130 Meilong Road, #268, Shanghai 200237, People's Republic of China.
authors.forename: Yuanhong
authors.initials: Y
authors.lastname: Zheng
authors.forename: Vanminh
authors.initials: V
authors.lastname: Le
authors.forename: Zhuoan
authors.initials: Z
authors.lastname: Cheng
authors.forename: Sheng
authors.initials: S
authors.lastname: Xie
authors.forename: Hegeng
authors.initials: H
authors.lastname: Li
authors.forename: Jianhui
authors.initials: J
authors.lastname: Tian
authors.forename: Jianwen
authors.initials: J
authors.lastname: Liu
mesh_terms: D001483:Base Sequence; D001943:Breast Neoplasms / Q000188:drug therapy; D002470:Cell Survival / Q000187:drug effects; D005260:Female; D017930:Genes, Reporter; D018840:HSP70 Heat-Shock Proteins / Q000235:genetics* / Q000378:metabolism; D006801:Humans; D007700:Kinetics; D008156:Luciferases / Q000235:genetics / Q000378:metabolism*; D061986:MCF-7 Cells; D008969:Molecular Sequence Data; D018384:Oxidative Stress / Q000187:drug effects*; D010778:Photochemotherapy; D017319:Photosensitizing Agents / Q000627:therapeutic use / Q000633:toxicity*; D011401:Promoter Regions, Genetic; D017382:Reactive Oxygen Species / Q000378:metabolism
chemical_list: D018840:HSP70 Heat-Shock Proteins; C490786:HSPA1A protein, human; D017319:Photosensitizing Agents; D017382:Reactive Oxygen Species; D008156:Luciferases