Abstract
Many cancer drugs are intended to kill cancer cells by inducing apoptosis. However, the potency assays used for measuring the bioactivity of these products are generally cell viability assays which do not distinguish between cell death and growth inhibition. Here we describe a cell-based fluorescence resonance energy transfer (FRET) biosensor designed to measure the bioactivity of apoptosis inducing cancer drugs. The biosensor contains cyan fluorescent protein (CFP) linked via caspase 3 and caspase 8 specific cleavage recognition sequences to yellow fluorescent protein (YFP). Upon caspase activation, as in the case of apoptosis induction, the linker is cleaved abolishing the cellular FRET signal. This assay closely reflects the mechanism of action of cancer drugs, in killing cancer cells and therefore can function as a potency test for different cancer drugs. We rigorously demonstrate this through characterization of a class of proteins targeting the death receptors. The one-step assay appears to be superior to other apoptosis-based assays because of its simplicity, convenience, and robustness.
Highlights
Potency is a measure of the activity of a drug in terms of concentration or amount required to produce a defined biological effect [1]
Generation of a cell-based FRET biosensor MB231 human breast cancer cells were stably transfected with an expression plasmid encoding cyan fluorescent protein (CFP)-linker-yellow fluorescent protein (YFP) fusion protein (MB231_CFP-YFP), wherein CFP functions as a donor and YFP as an acceptor for fluorescence resonance energy transfer (FRET)
We anticipate that given these advantages, the stably expressed FRET probe can be adapted for use in future relevant drug discovery and development studies
Summary
Potency is a measure of the activity of a drug in terms of concentration or amount required to produce a defined biological effect [1]. Many chemotherapeutic agents were identified by high throughput screening of compound libraries using cell viability assays with MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) or other related dyes [2,3]. This assay format has been widely adopted as a potency test in the development of cancer drugs. Cell viability assays cannot distinguish between cell death and growth arrest effects, leaving a caveat in the assessment of the true bioactivity of a cancer drug. As the goal of cancer therapy is to kill cancer cells, it is critical to assess the ability of a drug to induce cancer cell death. The form of cell death most commonly associated with cancer treatment, both in vivo and in vitro is apoptosis, or programmed cell death
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Free) 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call
