A high throughput drug screen based on fluorescence resonance energy transfer (FRET) for anticancer activity of compounds from herbal medicine

Abstract

Background and purpose: We report the development of a very efficient cell-based high throughput screening (HTS) method, which utilizes a novel bio-sensor that selectively detects apoptosis based on the fluorescence resonance energy transfer (FRET) technique. Experimental approach: We generated a stable HeLa cell line expressing a FRET-based bio-sensor protein. When cells undergo apoptosis, they activate a protease called 'caspase-3'. Activation of this enzyme will cleave our sensor protein and cause its fluorescence emission to shift from a wavelength of 535 nm (green) to 486 nm (blue). A decrease in the green/blue emission ratio thus gives a direct indication of apoptosis. The sensor cells are grown in 96-well plates. After addition of different chemical compounds to each well, a fluorescence profile can be measured at various time-points using a fluorescent plate reader. Compounds that can trigger apoptosis are potential candidates as anti-cancer drugs. Key results: This novel cell-based HTS method is highly effective in identifying anti-cancer compounds. It was very sensitive in detecting apoptosis induced by various known anti-cancer drugs. Further, this system detects apoptosis, but not necrosis, and is thus more useful than the conventional cell viability assays, such as those using MTT. Finally, we used this system to screen compounds, isolated from two plants used in Chinese medicine, and identified several effective compounds for inducing apoptosis. Conclusions and Implications: This FRET-based HTS method is a powerful tool for identifying anti-cancer compounds and can serve as a highly efficient platform for drug discovery.

A membrane‐bound FRET‐based caspase sensor for detection of apoptosis using fluorescence lifetime and total internal reflection microscopy

“…for the evaluation of drug effects on cells. Standard plate readers and flow cytometry have been used for the detection of FRET in cytoplasmically localized caspase sensors using ratio imaging (6,7). A high-throughput FRET screening system for larger cell numbers, which is based on TIRFM and x,y scanning of cells on a coverslip, has recently been reported (34).…”

Ready for a comeback of natural products in oncology

“…Beside cytotoxicity measurements, screening procedure for the discovery of novel NP frequently relies on the use of specific cell lines adapted or engineered to detect drug-induced apoptosis [55], cell cycle perturbations or alteration of specific molecular pathways. A typical example is that of the proteasome, a key component for limiting tumor cell proliferation and a target particularly well suited to the identification of NP.…”

Characterization of cytotoxic compound from mangrove derived fungi Irpex hydnoides VB4

“…The plotted mycelium was crushed using mortar and pestle with ethyl acetate and methanol and subjected to sonication (Sartorious Labsonic) for [3][4] h to obtain intracellular metabolites. Centrifuged at 2 000-2 500 rpm for 5 min and the supernatant were used for further studies.…”

“…Cancer is a leading cause of death worldwide [4,5] . As many anticancer drugs cannot discriminate cancer cells from non-cancer cells, many normal cells are also killed during the process of chemotherapy.…”