Development of rapid and highly sensitive HSPA1A promoter-driven luciferase reporter system for assessing oxidative stress associated with low-dose photodynamic therapy
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.
KeywordsPhotodynamic therapy (PDT) HSPA1A promoter activity Stress oxidative Luciferase reporter Cell viability
Heat shock protein
Reactive oxygen species
Micromoles per liter
This study was supported by the National Natural Science Foundation of China (NSFC project number: 81173224), the Shanghai Municipal Committee of Science and Technology(No. 11ZR1437000), and the Scholar of Longhua Hospital (D-11).
- Brondani Da Rocha A, Regner A, Grivicich I, Pretto Schunemann D, Diel C, Kovaleski G, Brunetto De Farias C, Mondadori E, Almeida L, Braga Filho A, Schwartsmann G (2004) Radioresistance is associated to increased Hsp70 content in human glioblastoma cell lines. Int J Oncol 25(3):777–785PubMedGoogle Scholar
- Demirovic D, Rattan SI (2012) Establishing cellular stress response profiles as biomarkers of homeodynamics, health and hormesis. Exp GerontolGoogle Scholar