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 regulatoryapproved 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.

Nutrient Analysis Critical Control Point (NACCP): Hazelnut as a Prototype of Nutrigenomic Study

“…CCL5 gene is one of several chemokine genes clustered on the q-arm of chromosome 17. In Yin-Ling Lin et al [36,37] paper, the authors suggested that the antioxidant used in their work effectively suppressed CCL5 mRNA expression levels, indicating that oxidant may be involved in the induction of the CCL5 gene expression by dengue-2-virus infection. In our work we found that the transcription levels of this genes were decreased (p ≤ 0.05) after hazelnut consumption.…”

Development of a multifunctional luciferase reporters system for assessing endoplasmic reticulum-targeting photosensitive compounds

“…The classical transcription mechanism of genes belonging to HSP family is inducing HSFs binding to specific HSEs in promoter of HSP genes (Liu et al 1993). It is widely documented PDT-mediated oxidative stress could activate HSF binding activity almost instantaneously after PDT treatment (Gomer et al 1996;Zheng et al 2013;Luna et al 2000). Furthermore, it has been reported that PDT treatment will induce thermotolerance in treated cells, and the activation of HSF binding activity after PDT is partly correlated with the induction of thermotolerance (Gomer et al 1996;Fuchs et al 2000).…”

“…PDT treatment PDT treatment was performed as described previously (Zheng et al 2013). Cells were co-incubated with certain concentrations of photosensitizers in the dark at 37°C.…”

“…After PDT treatment, DCFH-DA was added to the media at a final concentration of 10 μM and incubated for 30 min at 37°C. Then, the generation of ROS was measured by fluorescence intensity using EnSpire® Multimode Plate Reader (Perkin Elmer, Boston, MA, USA) at an excitation wavelength of 485 nm and an emission wavelength of 535 nm (Zheng et al 2013). …”

“…A recent study has shown that MCF-7 cells containing a human HSPA1A promoter-driven luciferase reporter could be used for assessing oxidative stress associated with low-dose photodynamic therapy (Zheng et al 2013). Our research developed cancer reporter cells expressing the firefly luciferase reporter under the control of the functional human GRP78 or CHOP promoter, respectively, and provided a comprehensive analysis of GRP78 and CHOP promoter-driven luciferase activity regulated by ER-targeting PDT.…”