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A Multiplexed Luciferase-based Screening Platform for Interrogating Cancer-associated Signal Transduction in Cultured Cells
Published on: July 3, 2013
Development of a multifunctional luciferase reporters system for assessing endoplasmic reticulum-targeting
Shengchao Lin1, Lingling Zhang, Kecheng Lei
1Department of Molecular & Cellular Pharmacology, Biomedical Nanotechnology Center, State Key Laboratory of Bioreactor Engineering & Shanghai Key Laboratory of New Drug Design, School of Pharmacy, East China University of Science and Technology, #268, 130 Meilong Road, Shanghai, 200237, People's Republic of China.
Abstract:
Photodynamic therapy (PDT) is a recently developed antitumor modality utilizing the generation of reactive oxygen species (ROS), through light irradiation of photosensitizers (PSs) localized in tumor. Interference with proper functioning of endoplasmic reticulum (ER) by ER-targeting PDT is a newly proposed strategy to achieve tumor cell death. The aim of this study is to establish a multifunctional model to screen and assess ER-targeting PSs based on luciferase reporters system. Upregulation of GRP78 is a biomarker for the onset of ER stress. CHOP is a key initiating player in ER stress-induced cell death. Here, the most sensitive fragments of GRP78 and CHOP promoters responding to ER-targeting PDT were mapped and cloned into pGL3-basic vector, forming -702/GRP78-Luc and -443/CHOP-Luc construct, respectively. We demonstrated that -702/GRP78-Luc expression can be used to indicate the ER-targeting of PSs, meanwhile estimate the ROS level induced by low-dose ER-targeting PDT. Moreover, the luciferase signaling of -443/CHOP-Luc showed highly consistence with apoptosis rate caused by ER-targeting PDT, suggesting that -443/CHOP-Luc can evaluate the antitumor properties of PSs. Hypericin, Foscan® and methylene blue were applied to verify the sensitivity and reliability of our model. These results proved that GRP78-CHOP model may be suitable to screen ER-targeting photosensitive compounds with lower cost and higher sensitivity than traditional ways.
Insights
This study introduces a new GRP78-CHOP model using luciferase reporters to screen endoplasmic reticulum-targeting photosensitizers for photodynamic therapy (PDT). This cost-effective model efficiently assesses photosensitizer targeting and antitumor efficacy.
Area of Science:
- Biochemistry
- Molecular Biology
- Oncology
Background:
- Photodynamic therapy (PDT) is an emerging antitumor treatment using photosensitizers (PSs) and light to generate reactive oxygen species (ROS).
- Targeting the endoplasmic reticulum (ER) with PDT offers a novel strategy for inducing tumor cell death.
- Biomarkers like GRP78 and CHOP indicate ER stress and ER stress-induced apoptosis, respectively.
Purpose of the Study:
- To develop and validate a multifunctional luciferase reporter model for screening and evaluating ER-targeting photosensitizers (PSs).
- To assess the model's ability to indicate PS localization to the ER, quantify ROS generation, and correlate with apoptosis rates.
Main Methods:
- Cloning of sensitive promoter fragments of GRP78 and CHOP into pGL3-basic vectors to create -702/GRP78-Luc and -443/CHOP-Luc constructs.
- Utilizing a luciferase reporter system to monitor gene expression in response to ER-targeting PDT.
- Validating the model's sensitivity and reliability using known PSs like Hypericin, Foscan®, and methylene blue.
Main Results:
- The -702/GRP78-Luc construct effectively indicates ER-targeting of PSs and estimates ROS levels from low-dose ER-targeting PDT.
- The -443/CHOP-Luc construct's luciferase signaling strongly correlates with apoptosis rates induced by ER-targeting PDT.
- The GRP78-CHOP model demonstrated high sensitivity and reliability in screening ER-targeting photosensitive compounds.
Conclusions:
- The developed GRP78-CHOP luciferase reporter model provides a sensitive and cost-effective platform for screening ER-targeting photosensitizers.
- This model can accurately assess PS ER-targeting, ROS generation, and antitumor efficacy, offering an advantage over traditional methods.
