Qi, blood, and cancer – What can we learn from the medicinal herb, Gynostemma pentaphyllum ( 絞股藍)

1. 2006 World Congress on Chinese Medicine Nov 23-25, 2006, Hong Kong Qi, blood, and cancer – What can we learn from the medicinal herb, Gynostemma pentaphyllum (絞股藍) W.L. Wendy Hsiao, Ph.D. School of Chinese Medicine Hong Kong Baptist University

2. What are the drug actions of anti-cancer Chinese herbal medicines? Kill cancer cells? Boost host immunity? Other effects?

3. A Story of Gynostemma pentaphyllum (jiao-gu-lan)

4. Materia Medica for Famine “救荒草本”明 永樂四年朱橚撰

5. Traditional applications Jiaogulan was first described as herbal medicine in 1578 A.D. by Li Shi-Zhen in his “Compendium of Materia Medica (本草綱木)” and he stated : “Jiaogulan is used to treat hematuria, edema and pain of the pharynx, heat and edema of the neck, tumors and trauma.” The TCM practitioner described it as “sweet, slightly bitter, neutral, warm, enhancing ‘Yin’ and supporting ‘Yang’ and suggested that it would be used to increase the resistance to infection and for anti-inflammation.”

6. Modern applications used as a sugar substitute for its sweetness. used as a medicinal herb for treating chronic bronchitis, hepatitis, high serum cholesterol, inflammation….  

7. The anti-cancer activities of Gp In vitro - reduce proliferation of certain tumor cell lines - induce apoptosis in human Hep3B and HA22T tumor cells In vivo - reduce the size of tumors - prolong lives of tumor-baring animals Clinical - reduce metastatic potential of primary tumors - alleviate side effects from chemo- and radiation therapy

8. Dammarane-type glycoside Anticancer Activities Dehydrated aerial parts In vitro cell model Animal study Modes of action Total Gp saponins Drug targets Specific pathways microarrays Proteomic study Protein fractionation Chemical analysis

9. . . . . . . . . . . . . . . . Focus formation assay GFP-Ras oncogene 2-3 weeks GFP-ras oncogene- Transformed cells Co-culture assay 7-10 days Normal cells (Rat 6)

10. Formation of a transformed GFP-ras-focus A B Day 1 C D Day 6 E F Day 20

11. Formation of GFP-ras-focus in the presence and absence of Gp saponins A B -Gp +Gp C D

12. Inhibitory effects of Gp require the presence of normal cells - Gp + Gp 103 GFP-ras cells 103 GFP-ras cells + 5x105 R6

13. Does the inhibitory effect of Gp require a direct contact to the normal R6 cells?

14. Gp effects on colony formation of GFP-ras cells in transwells with or without R6 cells 100 GFP-ras cells 100 GFP-ras cells + 105 R6 cells

15. Detection of a bioactive fraction from the conditioned medium from Gp-treated R6 cultures NH4SO4 precipitation normal conditioned medium Gp conditioned medium 25% 60% 80% 25% 60% 80% A B C A B C 1X 5X

16. Drug targets of Gp saponins

17. Ras-dependent Signaling Pathways Mitogens Stress ? Raf Raf Ras Rac1 PAK1 S218 P P MEKK1 Mek Mek S222 P P ? Rho X X SEK1 T183 P P P P MKK3 MKK3 MAPK1/2 MAPK1/2 P P Y185 PI3K RSK RSK P P p38 p38 P P JNK1 PIP2 MAPKAPK2 MAPKAPK2 P P P P Actin P P P P P P p38 p38 MAPK1/2 MAPK1/2 JNK1 RSK RSK P P P P P P P P P P P P P P ATF2 S63 P P JUN S73 Elk Elk - - 1 1 SRF SRF P P HIF1A c-myc

18. Gp saponins down regulate Raf-1 and alternate p-Erk1/2

19. Proteomic analysis

20. 2-D Image for cell lysates from Gp-treated and Untreated R6 cells - Gp + Gp WM pH10 pH10 pH 3 pH 3

21. 2-D Image for cell lysates from Gp-treated and Untreated R6 cells 90 K 70 K 60 K 50 K 30 K MW pH 4 pH 10

22. Up-regulated proteins Down-regulated proteins Enolase Beta-subunit (AA 1-312) Vimentin Mitochondrial aconitase Heat shock 70 protein 8 Heat shock 70 protein 5 Heat shock 60 protein 1 Heat shock 27 Pyruvate kinase Protein disulfide-isomerase Vimentin Aldehyde dehydrogenase Glutamate dehydrogenase Calreticulin  Enolase 1 Annexin 1 Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) ATP synthase  ATP synthase  Sestrin Rho GDI-1 Prohibitin

23. Up-regulated proteins Down-regulated proteins Enolase Beta-subunit (AA 1-312) Vimentin Glucose Mitochondrial aconitase Heat shock 70 protein 8 Heat shock 70 protein 5 Heat shock 60 protein 1 Heat shock 27 Pyruvate kinase Protein disulfide-isomerase Vimentin Aldehyde dehydrogenase Glutamate dehydrogenase Calreticulin  Enolase 1 Annexin 1 Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) ATP synthase  ATP synthase  Sestrin Rho GDI-1 Prohibitin Glycolysis (10 Rxs) 2 Pyruvate 2 Acetyl-CoA Citric Acid Cycle Oxidative phosphorylation ATP

24. Glucose Glucose Hexokinase1/2 Glucose 6-P Phosphoglucose isomerase Glycolysis (10 Rxs) Fructose 6-P Phosphofrutokinase 2 Pyruvate Fructose 1,6-P Aldolase Dihydroxyacetone P √ Triose Phosphate isomerase Myc HIF-1 Glyceraldehyde 3-P 2 Acetyl-CoA √ GAPDH √ 1,3-Bisphosphoglycerate Aldehyde DHase Glutamate DHase Citric Acid Cycle Phosphoglycerate kinase 3-Phosphoglycerate Phosphoglycerate mutase Oxidative phosphorylation 2-Phosphoglycerate √ Enolase √ Phosphoenolpyruvate ATP synthase  ATP synthase  √ Pyruvate kinase pyruvate ATP LDHase Lactate

25. Gp saponins downregulate HIF1A Western blots

26. Fatty food

27. *** ** # # Concomitant suppression of hyperlipidemia and hypercholesterol in Apcmin/+ mice by Gp p<0.05 #; p<0.01 **; p<0.001 ***

28. Acyl-CoA dehydrogenase Fatty acid metabolism Oxidation Fatty acid Acyl-CoA synthetase Fatty acyl-CoA thiolase

29. Cellular Functions & Growth ATP ATP ATP ATP HIF1A c-myc

30. Separation of Gp saponins by ODS column chromatograph Total Gp saponins Eluted with gradient CHCl3-MeOH silicon gel Fraction40-43 44-47 62-67 71-77 85-101 105-116 140-161 194-199 223-255 301-308 309-320 321-334 335-360 65-85% MeOH ODS column Single D A J K I G O N P Q V R U compounds B H W F Liu et al., J Nat Prod 2004 Liu et al., Planta Medica 2005

31. 1. GP-B 2. GP-G 3. GP-I 4. GP-P 5. GP-O 6. GP-Q 7. GP-N 8. GP-R2 9. GP-R 10.GP-W C-18 Column spectrum of total Gp saponins

32. Effect of pure Gp saponins on colony formation of the GFP-ras co-cultured with R6 Compounds Dosage ( µg/ml ) Inhibitory effect B 50 - 200 ++ G 50 + 200 ++++ H - 200 ++ I 50 - 200 +++ O 50 - 200 ++++ * Inhibitory effect: ++++, 100%; +++, 75%; ++, 50%; +, 25%; -, no effect.

33. Conclusions • The inhibitory effect of Gp requires the presence of co-cultivated normal cells. • Gp may render the co-cultivated normal cells secreting factors against the growth of transformed cells. (扶正抑邪) • Gp targets on Raf-1 and Erk1/2, the MAP kinase signaling molecules that play a crucial role in cancer development. • Proteomic analysis suggested that Gp saponins may play a crucial role in down-regulating cellular bioenergetics and catabolism, which may in turns suppress the growth of transformed cells. (清熱消腫)

34. Acknowledgments Hong Kong Baptist University Martin Mo Keith Wu William Tai Xin Liu University of Hong Kong Jen-Fu Chiu Qing-yu He

36. Cancer evolution is a multistage process Proposed molecular genetic events in the evolution of colon cancer p53 Hyper- proliferative epithelium Normal epithelium Early adenoma Late adenoma Carcinoma DNA hypermethylation APC K-ras mutation DCC deletion Cancer represents a complex interplay of abnormal genetic and epigenetic events

37. Chemoprevention: an essential approach to controlling cancer

38. Chemopreventive herbs - One in 4,000 Plants To be considered a true chemopreventive agent, a plant must conform to the following criteria: • The plant must be nontoxic and totally harmless to the body. • The action it exerts must be nonspecific and should maintain normal body functions despite a wide range of onslaughts to the body (i.e. stress). • It should normalize body functions irrespective of existing pathological conditions.

39. Objectives • To explore the anticancer effect of Gp saponins in R6/ras cell system • To characterize the anticancer effect of Gp saponins • To identify the drug targets of Gp saponins • To identify and purify bioactive components from total Gp saponins

40. Views of GFP/ras-transformed R6 cells under a fluorescence scope

41. What had happened to the oncogene-transfected cells? ? + Gp - Gp • Oncogene integraded, but failed to express. • Oncogene expressed, but could not transform R6 cells. • Oncogene intergraded cells failed to grow. Three possible modes of inhibition

42. Gp inhibitory effect is correlated with the number of the co-cultivated R6 cells in the transwell assay R6 cells2.5x105 2.5x104 2.5x103 -Gp +Gp 50 GFP-ras cells per transwell

43. 107 NM NM +Gp CM CM + Gp 106 No. of cells 105 104 103 days 0 2 4 6 8 Growth curves of the GFP-ras cells in normal and conditioned media derived from R6 cultures treated with or without Gp

44. The effect of size fractions of CM on colony formation Fresh medium CM - Gp CM + Gp MW 3000~ 10000 >3000 >12000

45. Medium---sample 1 CM – Gp (100µg) CM+Gp(100µg) 1 1 3 3 2 2 4 4 5 5

46. CM +Gp CM - Gp Area 2 60µg 100µg 100µg 100µg

47. CM+Gp CM Area 3 60µg 100µg 100µg 100µg

48. KD 108 90 50 35 28 21 PI3K

49. Western blotting for phosphatidylinositol 3-kinase p85- subunit (81 kD/pI: 5.8) in conditional medium Silver staining Western blotting PI3K McAB 1:1000 exp 4 hours

50. Signaling pathways downstream of PI3Kinase