Dr. Kathy Qian Luo Hong Kong University of Science & Technology Department of Chemical Engineering

1. A FRET-based high throughput drug screening method for discovery of anti-cancer compounds from traditional Chinese medicine Dr. Kathy Qian Luo Hong Kong University of Science & Technology Department of Chemical Engineering Bioengineering Graduate Program

2. Outline • Development of sensor C3 for detecting caspase-3 activation during apoptosis in living cells • Application of sensor C3 in screening anti-cancer drugs from traditional Chinese medicine

3. Apoptosis is closely related to the development of several severe diseases in human • Tissue homeostasis is tightly maintained by cell division and cell death. • Defects in apoptosis allow cells to over grow: • Cancer • Autoimmune diseases • Premature death in neuronal cells can cause: • Alzheimer’s disease • Parkinson’s disease

4. The signaling pathway of apoptosis Chemicals reagents e.g. Taxol, vincristine Irradiation (e.g. UV, x-ray, -ray, etc.) Death ligand (TNF or Fas) Bid Bax Bcl-2 tBid caspase-8 Mitochondria Smac Cyto c Apaf-1 IAP caspase-9 caspase-3 cell death

5. DEVD CFP YFP h1 h2 Caspase-3 cleavage DEVD h3 CFP h1 YFP Principle of a FRET (fluorescence resonance energy transfer) based bio-sensor in detecting caspase activation

6. DEVD CFP YFP h1 h2 Caspase-3 cleavage DEVD h3 CFP h1 YFP By measuring the fluorescence emission ratio between YFP and CFP, we can detect the activation of caspase-3 in living cells during apoptosis In normal cells, when the caspase-3 is not activated Em (YFP) Em (CFP) = HIGH In apoptotic cells, when the caspase-3 is activated Em (YFP) Em (CFP) = LOW

8. Seeding sensor cells Adding TCM compounds 1 2 3 Procedures of HeLa C3-based high throughput drug screening method Anti-cancer agents can change the color of sensor cells from green to blue 4 Detecting fluorescent signals using a plate reader and analyze the data using a computer

9. Anticancer drug, vinblastine (長春新鹼)can activate caspase-3 and induce apoptosis in HeLa-C3 cells

10. About 3-4 fold reductions in the emission ratio of YFP/CFP was detected by a fluorescent plate reader after cells were induced into apoptosis by multiple anti-cancer drugs

11. Application of sensor C3 to screen compounds isolated from Podophyllum emodi Wall. (桃儿七）

12. Podophyllotoxin related compounds

13. Comparison of 7 PD compounds at 20 nM

14. Podophyllotoxin related compounds

15. Deoxypodophyllotoxin (DP1) is more effective in activating caspase-3 than anti-cancer drug vincristine

16. Application of sensor C3 to screen compounds isolated from Salvia miltiorrhiza bunge (Danshen)(丹参) • Danshen has been used to treat hematological abnormalities, heart diseases, hepatitis and cancer. • Over 40 tanshinone compounds have been isolated, among them tanshinone IIA, tanshinone I cryptotanshinone, and dihydro-tanshinone are the major componants.

17. Tanshinone related compounds

19. Screening results of tanshinone compounds

20. Cytotoxicity effects of TIIA HeLa MCF-7 HepG2 HL60

21. Tanshinone IIA can prevent the growth of human breast tumor (MDA-MB-435) xenografts in nude mice

23. Summary • A highly efficient, cell-based, high throughput screening method for anti-cancer drug discovery has been established. • We have used this system to successfully screen over 80 herbal compoundsor extracts and found that its accuracy in determining apoptotic cell death is 100%. • Some of the newly identified herbal compounds have the potential for anti-cancer drug development. • One lead compound, tanshinone IIA, can induce apoptotic cell death in several cancer cell lines and prevent the growth of human breast tumor xenografts in nude mice.

24. Prof. Kathy Luo’s Lab Tian Hong lei Louisa Ip David Lv Jane Xu Naining Xu Pat Chung Kenneth Tsui Elizabeth Zhang Abraham Kwong Chan Chun Lai Pok Yui Cyril Lee Yu Yeung Dave Kwok Kwan Ting Paddy Prof. Donald Chang Yongmei Pu Vivian Yu Biology Department of HKUST Prof. Howei Luo Department of Natural Medical Chemistryat China Pharmaceutical University Nanjing, China Acknowledgements

25. Thank You!

29. Sensor C3 can be specifically cleaved by caspase-3

30. The FRET effects of sensor C3 is sensitive to caspase-3 cleavage Em of CFP=480 nm Em of YFP=526 nm

31. 1.1 0.9 0.7 Relative YFP/CFP ratio 0.5 0.3 0.1 0.01 0.10 1.00 10.00 100.00 caspase-3 [nM] A significant reduction of FRET effects was quickly generated when the sensor C3 was cleaved by the caspase-3 50% FRET reduction

32. Emission ratio of YFP/CFP and cell viability has a linear correlation when cells undergo apoptosis

33. Sensor C3 can distinguish cell death between apoptosis and necrosis

34. Deoxypodophyllotoxin (DP1) arrests sensor C3 cells in mitosis and induces apoptosis