Magnolol and Honokiol Augment Differentiation of Human Leukemia Cells Induced by

1. Magnolol and Honokiol Augment Differentiation of Human Leukemia Cells Induced by 1,25-Dihydroxyvitamin D3 and Retinoic acid (厚朴酚及和厚朴酚促進人白血病細包分化作用研究) Tse Kai Wing, Anfernee (Ph.D. student / CityU) Date: 2006-Nov-24 Supervisor: Prof. WF Fong

2. My Ph.D. Supervisor Professor FONG Wang-fun, David Director of the Research and Development Division School of Chinese Medicine Hong Kong Baptist University

3. Running title: Traditional Chinese Medicine Enhances Conventional Drug’s Pharmacological Effects

4. Background Problem addressed: Side effects of conventional drugs Strategy: Exploring potential combinational agent  Allow the use of lower dose of conventional drugs

5. Background Cancer model: Acute promyelocytic leukemia Conventional drugs: 1,25-dihydroxyvitamin D3 (VD3) All-trans retinoic acid (ATRA) Combination agents: Magnolol (厚朴酚) / Honokiol (和厚朴酚)

6. Nature of the Acute Promyelocytic Leukemia (APL)

7. General Method of Treatment of APL Myeloid Leukemia cells Apoptosis Apoptotic body Differentiation

8. Differentiating agents of APL Two differentiation inducing agents with no general cytotoxicity • All-trans retinoic acid (ATRA) • Granulocytic differentiation (Huang et al 1988, Blood;72:567) X  Relapse, drug resistance (Degos et al 1990, Lancet;336:1440) X  ATRA syndrome (Frankel et al 1992, Ann Intern Med;117:292) 1,25-dihydroxyvitamin D3 (VD3)  Monocytic differentiation (McCarthy et al 1983, Leuk Res;7:51) X  Systemic hypercalcemia

9. Differentiating agents of APL All-trans retinoic acid (ATRA) 1,25-dihydroxyvitamin D3 (VD3)

10. Differentiating agents of APL Strategy deal with side effects of ATRA and VD3 • Design structural analogs based on low calcemic activity, eg. ZK 191784 (Zugel et al 2002, J Invest Dermatol 119:1434) • Tissue selective VDR modulators (VDRM) • Use of VD3 in combination with non-toxic doses (natural compounds) which enhances the sensitivity of APL cells to VD3 this study

11. Hypothesis From literature: agents withanti-inflammation propertiescan enhance the terminal differentiation of induced leukemia cells (Sokoloski JA, etal. Leuk Res. 22(2):153-61 and Oncol Res. 9(1):31-9) Hypothesis: crude extracts of TCM and its activecomponents withanti-inflammation propertieswould produce an enhancing effect on VD3/ATRA-induced HL-60 cell differentiation.

12. Active compounds in Magnolia officinalis (厚朴) Magnolol 厚朴酚 Honokiol 和厚朴酚 厚朴 Magnolia officinalis

13. Combined Effects of Magnolol(MG) and Honokiol(HK) with 1nM VD3 on HL-60 cells * * * * * for MG/HKV1 > MG/HK + V1, p<0.01 (n=3)

14. Combined Effects of Magnolol(MG) and Honokiol(HK) with 20nM ATRA on HL-60 cells * # * for MG/HKV1 > MG/HK + V1, p<0.01 (n=3) # for MG/HKV1 > MG/HK + V1, p<0.05 (n=3)

15. Combined Effects of Magnolol(MG) and Honokiol(HK) with VD3 or ATRA on HL-60 cells • Potentiation also observed in nitro-blue tetrazolium reduction assay • Cell cycle – cell cycle block in G1 phase • Increase in cyclin-dependent kinase inhibitor p27Kip • MG/HK alone or combination did not induce apoptosis • Inhibition of extracellular signal-regulated kinase (ERK) blocked the differentiation enhancement induced by MG/HK • Magnolol and honokiol cooperatively enhance cell differentiation induced by VD3 and ATRA. • MG and HK enhanced the cytotoxic effects of VD3 in VDR-positive cells MCF-7 but not in VDR-negative MDA-MB-231 cells

16. Publication: Magnolol and honokiol enhance HL-60 human leukemia cell differentiation induced by 1,25-dihydroxyvitamin D3 and retinoic acid. Int J Biochem Cell Biol. 2005 Feb;37(2):427-41.

17. Mechanistic Studies

18. Action of Vitamin D Receptor on VDRE p65 • Lu X etal. Integration of the NfkappaB p65 subunit into the vitamin D receptor transcriptional complex: identification of p65 domains that inhibit 1,25-dihydroxyvitamin D3-stimulated transcription. (J Cell Biochem. 2004 Jul 1;92(4):833-48.) •  NF-B is constitutively activated in human myelogenous leukemia cells including HL-60 cells (Baumgartner et al., 2002; Frankenberger et al.,1994; Griffin, 2001; Guzman et al., 2001).

19. Transcription factor – NF-κB Nuclear Factor kappa B (NF-κB): first described by Sen and Baltimore (1986) Transcriptional Activity DNA binding Heterodimer of NF-κB (p65/p50)

20. NF-κB Activation Pathway Stimulus, eg. TNF-α, PMA, LPS IKK

21. NF-κB Inactivation by active compounds from TCM IKK activation 薑黃素(Curcumin) IkB Phosphorylation 大黃素 (Emodin) Proteosome inhibitor 兒茶素(EGCG) p65/p50 – DNA binding 冬凌草甲素(Oridonin)

22. Further studies: Honokiol inhibits TNF-alpha-stimulated NF-kappaB activation and NF-kappaB-regulated gene expression through suppression of IKK activation. Biochem Pharmacol. 2005 Nov 15;70(10):1443-57. Magnolol inhibits TNF-alpha-stimulated NF-kappaB activation and NF-kappaB-regulated gene expression through suppression of IKK activation. submitted

23. NF-κB Inactivation by MG / HK IKK inactivation MG/HK

24. Future studies: Role of transcription factor NF-kB in vitamin D receptor function and cancer drug discovery CERG Grant 2007

25. Summary: • Magnolol (厚朴酚) / Honokiol (和厚朴酚) are potential differentiation enhancing agents which may allow the use of low doses of VD3 and ATRA in the treatment for acute promyelocytic leukemia. • provide a scientific basis for future clinical testing of various combinations of steroid hormone ligand (eg.VD3) and NF-κB antagonists in cancer differentiation therapy. • Explore the use of TCM as enhancing agents to minimize the side effects of conventional drugs

26. Thank you for your attention !!!