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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.

Oblongifolin C (OC) was identified as a potent apoptosis inducer from an herbal plant, Garcinia yunnanensis, during our previous bioassay-guided drug screening. In this study, we investigated the signaling pathways through which OC activated apoptosis in HeLa cells. We also compared the IC 50 values of OC with that of etoposide, paclitaxel and vinblastine in multiple cancer cell lines including HER2 and P-glycoprotein overexpressing cells. In addition, the in vivo antitumor effect of OC was studied in nude mice model. Our results showed that OC induced a caspase-dependent apoptosis by triggering a series of events in HeLa cells including Bax translocation, cytochrome c release, caspase-3 activation, chromosome fragmentation followed by caspase-8 activation, Bid cleavage and eventually cell death. Addition of a pan-caspase inhibitor or overexpression of an anti-apoptotic protein, Bcl-xL, prevented OC-induced cell death. Moreover, OC exhibited a wide anticancer spectrum in multiple cancer cell lines with comparable IC 50 values, regardless of the expression levels of HER2 and P-glycoprotein. In contrast, the IC 50 values of three clinical anticancer drugs, etoposide, paclitaxel and vinblastine were significantly elevated in HER2 and/or P-glycoprotein overexpressing cells. Furthermore, OC showed a similar antitumor effect but lower general toxicity than etoposide against xenografted human tumors in nude mice model. All these data suggested that OC is a promising apoptosis inducer with the potential to be developed into a clinical anticancer drug.

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In order to improve the comprehensive utilization of major by-products in apple-juice processing, the components, antioxidant and antimicrobial activities of oil in two species apple seeds, Fuji and New Red Star, were investigated. The Soxhlet extracted oil content of apple seeds raged from 20.69 to 24.32 g/100 g. The protein, fiber and ash contents were found to be 38.85-49.55 g/100 g, 3.92-4.32 g/100 g and 4.31-5.20 g/100 g, respectively; the extracted oils exhibited an iodine value of 94.14-101.15 g I/100 g oil; refractive index (40 degrees C) was 1.465-1.466; density (25 degrees C) was 0.902-0.903 mg/ml; saponification value was 179.01-197.25 mg KOH/g oil; and the acid value was 4.036-4.323 mg KOH/g oil. The apple seed oils mainly consisted of linoleic acid (50.7-51.4 g/100 g) and oleic acid (37.49-38.55 g/100 g). Other prominent fatty acids were palmitic acid (6.51-6.60 g/100 g), stearic acid (1.75-1.96 g/100 g) and arachidic acid (1.49-1.54 g/100 g). Apple seed oil was proven to possess interesting properties, emerging from its chemical composition and from the evaluation of its in vitro biological activities. The apple seed oil was almost completely active against bacteria, mildews were less sensitive to apple seed oil than yeasts, and the minimum inhibitory concentration (MIC) of apple seed oil ranged from 0.3 to 0.6 mg/ml. The observed biological activities showed that the oil had a good potential for use in the food industry and pharmacy.

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