Zhi-xiu Lin PhD* , Lau Sin Ting Cynthia* & Po-Sing Leung PhD #

1. Brucea javanica fruit-mediated cytotoxicity on cultured pancreatic cancer cells is via induction of apoptosis Zhi-xiu Lin PhD*, Lau Sin Ting Cynthia* & Po-Sing Leung PhD# *School of Chinese Medicine and #Department of Physiology The Chinese University of Hong Kong

2. Pancreatic Cancer Statistics • 4th, 5th & 10th leading cause of cancer deaths in U.S., China & HK, respectively • 6 times increase in the reported incidence in China over the last 20 years • Usually diagnosed at advanced stage • Propensity to metastasize • Intrinsic resistance to radiotherapy and chemotherapy

3. Pancreatic Cancer Statistics • Annual mortality rate ≈ Annual incidence rate • 1-yr overall survival rate = 12% • 5-yr overall survival rate < 5% • “King of Cancers!”

4. Pathology of Pancreatic Cancer • Originate from exocrine and endocrine pancreas • ~90% in the exocrine portion • >85% are classified as ductal adenocarcinoma

5. Exocrine pancreas Endocrine pancreas Hormones production e.g. Insulin, glucagon Digestive enzyme production Pancreas Ductal Adenocarcinoma Pancreatic Neuroendocrine Tumours (PNETs) Types of Pancreatic Cancer

6. High risk people for Pancreatic cancer Risk Factors Chronic pancreatitis & • Diabetes mellitus

7. Treatments of Pancreatic Cancer • Surgery • Remains the most effective treatment • However, only suitable for early stage patients (~10-20%) • Local recurrences • Chemotherapy • Gemcitabine–based treatment • Approved as standard first-line chemotherapeutic treatment by FDA in 1995 • For unresectable and advanced stage • Improvement in quality of life • Does not reduce the size of the tumor • Radiotherapy Gemcitabine

8. Resistance of Pancreatic Cancer to Cell Death • Pancreatic cancer is highly resistant to apoptotic stimuli, because • Overexpression of growth factors • Epidermal growth factor • Insulin-like growth factor-1 • TGF-α • Fibroblast growth factor and their receptors • TGF-βisoforms • Diminished cytochrome C release • Constitutive active NFκB activity • Bcl-2 expression determine progression and survival • Mutation of p53

9. Chinese Medicine for Cancer Treatment • Chinese medicine as an alternative and complementary treatment to Pancreatic cancer • Improve clinical symptoms • Enhance quality of life • Delay cancer recurrence and metastasis • Prolong patient’s survival You et al. 2000; Yang et al. 2001; Yang et al. 2002

10. Aims of Study - • To investigate the cytotoxic effects of nine common anti-cancer Chinese medicinal substances on pancreatic cancer cell lines • To evaluate the apoptotic activity of the most potent anti-pancreatic cancer herb (Brucea javanica fruit)

11. ground 100g + 1L 80% ethanol Reflux 2-hr Storage at -20oC Chinese medicinal materials & extracts preparation • 9 Chinese medicinal substances were selected for the study based on ethnomedical evidence

12. 9 Chinese medicinal substances

13. Pancreatic Cancer Lines & Culture Conditions • Panc-1 • Organ: pancreas • Tissue: duct • Disease: epithelioid carcinoma • DMEM medium, 10%FBS, 4mM L-glutamine, 100U/ml PS, 5% CO2 • SW-1990 • Organ: pancreas • Disease: adenocarcinoma • Derived from metastatic site: spleen • L-15 medium, 10% FBS, 2mM L-glutamine, 100U/ml PS, free gas exchange atmospheric air • Capan-1 • Organ: pancreas • Disease: adenocarcinoma • Derived from metastatic site: liver • IMDM medium, 20% FBS, 4mM L-glutamine, 100U/ml PS, 5% CO2

14. Nontumorigenic Cell Line & Culture Conditions • Hs68 • Organ: foreskin • Cell type: fibroblast • DMEM medium, 10%FBS, 4mM L-glutamine, 100U/ml PS, 5% CO2

15. Assessment of Cytotoxicity • Sulphorhodamine B (SRB) assay • Detection of viable cells • Water soluble dye • Bind basic amino acid of cellular proteins • Total protein mass is related to cell number

16. SRB ASSAY DRUG 72 hr incubation 4 hr Pre-incubation 1 hr at 4oC Air Dry Remove supernatant Wash 4 times with nano-H2O 25μl ice-cold 10% Trichloroacetic Acid (TCA) 100μl 0.4 % (w/v) SRB Measure A550nm Wash 4 times with 1% acetic acid Air Dry 150μl Tris-Base

17. Relative Growth Percentage (%) concentration (μg/mL) The cytotoxic effects of four chemotherapeutic drugs on Panc-1. Cells were treated with increasing concentrations of desired drugs for 3 days. 5000 cells per well were seeded in 96-well plates. The viabilities of cells were determined by SRB ( ) and MTT ( ) assay. Data are expressed as percentage (± SEM) of the control cells (medium alone). Each point represents a mean of at least 3 independent experiments. Cytotoxicity of 4 chemotherapeutic agents on Panc-1 cells

18. Cytotoxicity of 4 Chemotherapeutic Agents *IC50 calculated using GraphPad Prism programme

19. Cytotoxic effects of Chinese herbal extracts on Panc-1 The inhibitory effects of the nine Chinese herbal extracts on Panc-1 cell determined by SRB assay. 5000 cells per well were seeded in 96-well plates. Cells were exposed to increasing concentrations of herbal extract for 3 days.

20. Cytotoxicity of 9 Chinese Medicinal Extracts *IC50 calculated using GraphPad Prism programme

21. Evaluation of B. javanica Fruit-Mediated Apoptosis • Cell Death Detection ELISAplus kit (Roche Applied Science) • Quantification of histone-complexed DNA fragments • Mouse monoclonal antibodies detect against DNA and histone • Western Blotting • Detecting Caspase-3 expression

22. Apoptotic effects of B. javanica fruit extract, camptothecin and gemcitabine on Panc-1 cells *P < 0.05 ** P < 0.01

23. Apoptotic effects of B. javanica fruit extract, camptothecin and gemcitabine on SW-1990 cells *P < 0.05 ** P < 0.01

24. BJ – 30μg/ml BJ – 1μg/ml BJ – 3μg/ml Control Caspase – 3 (20kDa) β- actin (42kDa) Western Blotting *P < 0.05

25. Cytotoxicity of different fractions of B. javanica extract *P < 0.01

26. Cytotoxicity of Bruceine D in Pancreatic Cancer Cell Lines Bruceine D *P < 0.01

27. Cytotoxicity of various agents to Hs68

28. A Comparison of cytotoxic Potency

29. Conclusions • B. javanica fruit possesses significant cytotoxicity on 3 common pancreatic cancer cell lines. • Induction of cellular apoptosis is, at least in part, responsible for the observed B. javanica-mediated cytotoxicity.

30. Future Work . . . • To study the effects of B. javanica extract on the cell cycle of pancreatic cell lines • To isolate more compounds from B. javanica for SAR study • To investigate the anti-angiogenic effect of B. javanica fruit and its compounds

31. Acknowledgements • Direct Grant, the Chinese University of Hong Kong (project no. 2030326, awarded to ZXL and PSL), and • Competitive Earmarked Research Grant from the Research Grants Council of Hong Kong (project no. CUHK4537/05M, awarded to PSL).

32. The Chinese University of Hong Kong Thank you for your attention!