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In Vitro Studies on the Growth-inhibitory and Differentiation-inducing Activities of 18 b -Glycyrrhetinic Acid on Neuroblastoma Cells. bbb. Lee, KW, Lo, FH , Mak, NK, Leung, KN Department of Biochemistry The Chinese University of Hong Kong Hong Kong, China.

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  1. In Vitro Studies on the Growth-inhibitory and Differentiation-inducing Activities of 18b-Glycyrrhetinic Acid on Neuroblastoma Cells bbb Lee, KW, Lo, FH, Mak, NK, Leung, KN Department of Biochemistry The Chinese University of Hong Kong Hong Kong, China

  2. Licorice (Radix Glycyrrhizae ) and its Dried Root (Gancao 甘草) • In China: Tonic (滋补), antipyretic(利尿), antidote (解毒), demulcent (镇痛),and expectorant (化痰), etc • In the West: Sweetening and flavouring agents

  3. Major Components of Licorice Root • - Glycyrrhizin (GL) (also called Glycyrrhizic acid) • A pentacyclic triterpenoid - Other components: • Essential oils • Alkaloids • Polysaccharides • Polyamines • Flavonoids

  4. Chemical Structures and Metabolism of the Major Components of Licorice Root Bacterial -D-glucuronidase in intestine 18-Glycyrrhetinic Acid (18-GA) Glycyrrhizin (GL) Major metabolite

  5. GL and 18-GA Anti-viral Anti-tumor Hepatoprotective Anti-inflammatory Immunomodulatory Pharmacological Activities of GL and GA

  6. Anti-tumor Activities of 18-Glycyrrhetinic Acid on Neuroblastoma Cells

  7. What is Neuroblastoma? • the most common extracranial childhood solid tumor • a tumor of the sympathetic nervous system that accounts for 8-10% of the pediatric tumors • development of human neuroblastoma is due to an arrest in differentiation program of neural crest progenitor cells

  8. Neuroblastoma Cell Lines Used in Our Study • Neuro-2a (subclone BU-1): a well established and subcloned cell line derived from a spontaneous neuroblastoma of a strain A albino mouse • SK-N-DZ: a human neuroblastoma cell line derived from a bone marrow metastasis from a child with poorly differentiated embryonal neuroblastoma • SH-SY5Y: a thrice cloned subline of the human neuroblastoma cell line SK-N-SH which was established from a metastatic bone tumor

  9. Characteristics of Neuroblastoma Cells • capable of unlimited proliferationin vitro • retain their ability of differentiation into neuronal cell types upon treatment with differentiating agents • usually employed as model systems to study the signals and mechanisms governing the division and differentiation of neuronal cells

  10. IC50 ~ 75 M IC50 ~ 97 M Anti-proliferative Effect of 18b-GA on Different Neuroblastoma Cell Lines IC50 ~ 93 M % Inhibition of 3H-TdR Incorporation Concentration of 18b-GA ( M)

  11. Kinetic Study on the Anti-proliferative Effect of 18b-GA on BU-1 Cells % Inhibition of 3H-TdR Incorporation Concentration of 18b-GA (M)

  12. Effect of 18b-GA on the Proliferative Potential of BU-1 Cells f = 1 in 7.9 f = 1 in 1.9

  13. Effect of 18b-GA on the Colony-forming Ability of BU-1 Cells in Soft Agar * * * * *p < 0.01

  14. Antiproliferative effect due to cell cycle disturbance??

  15. DMSO control G1: 40.95% S: 44.33% G2: 14.72% 80M GA G1: 21.88% S: 35.87% G2: 42.25% 100M 18b-GA DMSO control G1: 40.95% S: 44.33% G2: 14.72% 100M GA G1: 31.14% S: 12.06% G2: 56.80% Cell Cycle Analysis of BU-1 Cells after Treatment with 18b-GA for 24 Hours 80M 18b-GA

  16. DMSO control G1: 41.34% S: 52.39% G2: 6.27% 80 M GA G1: 14.46% S: 26.25% G2: 59.28% 80M 18b-GA DMSO control G1: 41.34% S: 52.39% G2: 6.27% 100 M GA G1: 27.70% S: 11.89% G2: 60.41% Cell Cycle Analysis of BU-1 Cells after Treatment with 18-GA for 48 Hours 100 M 18b-GA

  17. Anti-proliferation G2/M Cell Cycle Arrest

  18. Cell Cycle Regulatory Proteins

  19. Effect of 18b-GA on the Cell Cycle Regulatory Genes Expression 8 hr 24 hr DMSO 80 100 120 DMSO 80 100 120 M Cyclin A (399 bp) (22 cycles) 2.91 1.91 1.26 0.70 2.47 2.08 1.50 0.71 Cyclin B (584 bp) (22 cycles) 2.14 1.49 0.98 0.49 1.92 1.61 1.23 0.61 Cdc2 (311 bp) (21 cycles) 2.69 2.06 1.63 1.31 2.89 2.65 2.32 1.53 GAPDH (226 bp) (20 cycles)

  20. Effect of 18b-GA on Cell Cycle Regulatory Proteins Expression 24 hr 48 hr DMSO 80 100 120 M DMSO 80 100 120 Cyclin A 15.74 10.38 11.14 5.12 12.87 11.98 9.17 6.50 Cyclin B 5.46 6.55 1.91 0.71 7.30 5.95 2.59 0.00 Cdc2 12.54 13.13 11.64 11.26 17.03 15.12 12.76 7.23 -Actin

  21. Conclusions (I) • 18b-GA exhibited growth-inhibitoryeffect on murine and human neuroblastoma cells in a dose- and time-dependent manner • 18b-GA reduced the proliferative potential on the BU-1 cells • 18b-GA suppressed the colony-forming ability of the BU-1 cells

  22. Conclusions (II) • 18b-GA impaired the cell cycle progression of BU-1 cells and caused the accumulation of cells in the G2/M phase • The cell cycle arrest could be mediated through the transcriptional and translational down regulation of cyclin A, cyclin B and cdc2

  23. Morphological Changes in BU-1 Cells after 3 Days of Treatment with 18b-GA DMSO control 60M 18b-GA 80 M 18b-GA 100 M 18b-GA

  24. Enhanced Expression of Neuronal Differentiation Markers after treatment with 18b-GA MAP2 Neurofilament-200 DMSO control 80 M 18b-GA

  25. Enhanced Acetylcholine Esterase (AChE) Activity after Treatment with 18b-GA ** *** Fold change in AChE activity Concentration of 18b-GA (M) **p>0.01; ***p>0.005

  26. 8 hr 24 hr DMSO 80 100 120 M DMSO 80 100 120 N-Myc (763 bp) (28 cycles) 75% 21% 20% 53% 55% 2.49 2.63 1.31 1.37 2.16 1.62 0.46 0.43 c-Myc (501 bp) (24 cycles) 1.60 1.61 1.44 1.37 1.28 1.42 1.48 1.57 GAPDH (226 bp) (20 cycles) Effect of 18b-GA on Proto-oncogene Expression

  27. 64% 73% Effect of 18b-GA on Differentiation-Related Genes Expression Ctl 30 60 120 240 360 480 min c-Fos (247 bp) (28 cycles) 0.73 1.22 2.57 1.84 1.49 1.18 0.88 c-Jun (459 bp) (26 cycles) 1.54 1.78 1.78 1.58 1.72 1.63 1.51 NF- B/p50 (250 bp) (28 cycles) 1.88 2.43 2.73 2.59 2.71 2.78 3.08 Bcl-2(293 bp) (27 cycles) 1.08 1.42 1.51 1.62 1.69 1.71 1.87 GAPDH(226 bp) (20 cycles)

  28. Conclusions (III) • 18b-GA inducedmorphological, phenotypical and functional differentiation inBU-1 cells • The differentiation of BU-1 cells induced by 18b-GA was accompanied with a down-regulation of N-myc gene expression and an up-regulation of the expression of the Bcl-2,NF-B/p50 and c-fos genes

  29. Future Perspectives • Elucidation of downstream signaling pathway(s) in 18-induced neuronal cell differentiation • Studies of the therapeutic potential of 18-GA on human neurological tumors using nude mouse models

  30. THE END

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