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.

Figure 1

The fluorescence emitted from HeLa-C3 cells changes from green to blue in response to induction of apoptosis. (a) HeLa-C3 cells grown on a cover-slip-based observation chamber were treated with three apoptotic inducers: 100 nM paclitaxel for 24 h, 10 ng ml⁻¹ TNF-α plus 10 μg ml⁻¹cycloheximide for 12 h and 12 h after UV-irradiation for 5 min. The phase images and fluorescent images of YFP and CFP were recorded from the same observation field in the green and blue channels, respectively. Merged color images of YFP (green) and CFP (blue) of HeLa-C3 cells show a significant increase in blue color exclusively in cells with cell shrinkage morphologies. The phase images show cell shrinkage and apoptotic bodies, which are significant indicators of apoptosis. Bar, 25 μm. (b) HeLa-C3 cells grown in a 96-well plate were treated with UV-irradiation (UV) for 5 min in the absence or presence of the pan caspase inhibitor Z-VAD-FMK (20 μM), or treated with 100 nM of paclitaxel (Tax) for 18 and 36 h. At the indicated time points, the fluorescence intensity was measured using a fluorescent plate reader. Data shown are means±s.d. from three experiments. (c) Caspase-3 activity was measured from HeLa-C3 cells using an in vitro assay described in Methods. The decrease in the Y/C emission ratio of the HeLa-C3 cells correlates well with the increase of caspase-3 activity during apoptosis. Data shown are means±s.d. from three experiments.

Figure 2

The CFP-DEVD-YFP fusion protein in HeLa-C3 cells is specifically cleaved during apoptosis but not necrosis. HeLa-C3 cells were treated with either an apoptotic inducer (5 min UV-irradiation) or a necrotic stimulus (5 mM H₂O₂) for various times and analyzed by Western blotting (a) and agarose gel electrophoresis (b). The cleavage of sensor protein was observed only during UV-induced apoptosis as indicated by caspase-3 activation (a) and DNA fragmentation (b). (c) HeLa-C3 cells grown in a 96-well plate were irradiated with 5 min UV light and then cultured for 9 h or treated with 5 mM H₂O₂ for 8 h. After detecting the fluorescence intensity of the cells using a fluorescent plate reader, cell viability was determined from the same sample well by MTT assay. Data shown are means±s.d. from three experiments.

Figure 3

A significant reduction in the Y/C emission ratio can be seen from HeLa-C3 cells treated with different anticancer drugs: paclitaxel (a), vincristine (b), ET (c) and hydroxyurea (d). A summary of FRET effects in HeLa-C3 cells in response to multiple anticancer drugs at their optimal concentrations (e). The error bars represent the s.d. from three independent experiments.

Figure 4

A clear correlation between a reduction in Y/C emission ratios and a decrease in cell viability is exhibited in cells treated with apoptotic stimuli but not in cells treated with necrotic inducers. HeLa-C3 cells grown in 96-well plates were treated with four different apoptotic inducers: 50 nM paclitaxel for 12–48 h, 50 ng ml⁻¹ nocodazole for 12–48 h, 100 μM ET for 12–48 h and 3–12 h after UV-irradiation for 5 min, and with four different necrotic inducers: H₂O₂ at 3, 5 and 7 mM for 2–8 h, 0.01 and 0.1% NP40 for 0.5 h, 0.1% Triton X-100 for 0.5 h and double-distilled H₂O for 0.5 h. The fluorescence intensity and cell viability of HeLa-C3 cells from different treatments were obtained in similar ways as described in Figure 2c. Data shown are from a single representative experiment.

Figure 5

Molecular structures of tanshinone family compounds.

Figure 6

Screening results of tanshinone family compounds. HeLa-C3 cells grown in 96-well plates were treated with six structurally related compounds of the tanshinone family at three concentrations (5, 10, 20 μM) for up to 48 h. The fluorescence intensities of YFP and CFP were detected separately using a fluorescent plate reader. The graphs show the efficacy of each compound in activating caspase-3 in HeLa-C3 cells. (a) Crypto, (b) MTQ, (c) TanNa, (d)TI, (e) TIIA, (f) TIIB. (g) Results of Y/C emission ratio levels at all three concentrations after HeLa-C3 cells were treated with six tanshinone family compounds for 48 h. Error bars in (a–f) represent s.d. from three independent experiments and those in (g) represent the s.e.m. from seven independent experiments.

Figure 7

Molecular structures of podophyllotoxin family compounds.

Figure 8

Screening results of S1 family compounds. HeLa-C3 cells grown in 96-well plates were treated with six podophyllotoxin family compounds plus ET at 10 nM (a), 20 nM (b) and 40 nM (c) for up to 48 h. Changes in the Y/C emission ratio were measured and used to compare the efficacy of each compound in inducing caspase-3 dependent apoptosis. (d) Apoptotic effects of DP1 and S1 were directly compared with the microtubule interfering agents nocodazole and vincristine, each at a concentration of 10 nM. (e) HeLa cells were treated with 10 and 20 nM S1 and 10 nM DP1 for up to 48 h. Phase images showing mitotic arrested cells with rounded cell morphology and apoptotic cells with cell shrinkage. Cells treated with 10 nM DP1 were also stained with Hoechst 33342, a DNA dye. The fluorescent images in the enlarged pictures show that 10 nM DP1 arrested cells in prometaphase at 12 h and caused DNA fragmentation after 24 h of drug treatment. The scale bars in the pictures of phase contrast and Hoechst staining are 50 and 25 μm, respectively. The error bars represent s.d. from three independent experiments.