Presentation (saiki) (5)

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Presentation (saiki) (5)

  1. 1. Anti-metastatic effect of intestinal bacterialmetabolites of ginseng saponins and their molecular mechanism of action University of Toyama Ikuo SAIKI, Executive Vice PresidentBiological activities of saponins extracted from the root of Panax ginseng C. A. MEYER • anti-diabetic effect • anti-hyperlipidemic effect • management of psychosomatic diseases (indefinite complaints, stress ulcer, anxiety neurosis) • inhibition of tumor-induced angiogenesis • anti-tumor activity • alleviation of some types of inflammatory diseases 21 Glc 1- 6 Glc-O 22 24 26 Glc-O OH 20 25 OH 23 11 12 13 17 27 19 18 16 1 14 2 9 10 8 15 3 30 4 5 6 7 HO Glc 1 -6 Glc -O 28 29 H O- Glc Protopanaxadiol-type Protopanaxatriol-type
  2. 2. Isolation and chemical structure of ginsenosides antheir intestinal bacterial metabolites White Ginseng 21 Glc 1- 6 Glc-O 22 24 26 Arap 1-6 Glc -O Araf - Glc -O Glc-O Glc -O OH OH OH OH OH 20 23 25 11 12 13 17 27 1 19 18 14 16 2 9 10 8 15 3 30 4 5 6 7 O Glc 1-6 Glc -O HO HO Glc 1 -6 Glc -O Glc 1 -6 Glc - 2 1 28 29 H H H O- Glc O- G lc - Rha Ginsenoside Rb1 Rb2 Rc Ginsenoside Rg1 Re Protopanaxadiol-type Protopanaxatriol-type Red Ginseng (Mild-acid treatment) Ginsenoside Rg3 Hydrolysis by Intestinal bacteria Glc -O OH Ginsenoside Rh1 Ginsenoside Rh2 HO HO H OH Compound K or M1 20(S)-protopanaxatriol! 20(S)-protopanaxadiol HO OH or M4Effect of oral or i.v. administration of ginsenosides and their metabolites on lung metastasis of B16-BL6 cells p.o. Intestinal Bacteria i.v.
  3. 3. Anti-metastatic effect by oral administration of ginsenosides may be primarily mediated by their intestinal bacterial metabolites (M1 or M4) Glc -O OH Intestinal bacteria M1 M1 HO H Protopanaxadiol-type and M4 HO OH Protopanaxatriol-type ginsenosides (Rb1, Rb2, Rc, Rg1, Re) M4 M1 M4 HO OH Lung metastasis Oral administration Ginsenoside Rb1 M1 ! Inhibition ! B16-BL6 !"#$% Metabolite M1 M1 ! Inhibition Intravenous administration Ginsenoside Rb1 " Metabolite M1 M1 ! Inhibition Time course of Rb1 and/or M1 level in the serumobtained from mice administered orally with Rb1 or M1 Rb1 M1 M1 M1 M1 M1 M1 M1 M1 Concentration in blood (&g/ml) 12 M1 10 : M1 8 : Rb1 6 4 2 0 0 !2 4 8 16 24 0 !2 4 8 16 24 Hours after oral administration
  4. 4. Relationship between parent and litters in Rb1(1 transformation rate# Intestinal bacteria Rb1 M1 low high 25 Parent Intestinal bacteria 0-20% 20-50% 50-70% 20Number of mice Litters 15 Rb1 M1 transformation rate (%) (at 6 week-old) 10 5 0 0- 10- 20- 30- 40- 50- 60- 70- 80- 0- 10- 20- 30- 40- 50- 60- 70- 80- 0- 10- 20- 30- 40- 50- 60- 70- 80- 10 20 30 40 50 60 70 80 90 10 20 30 40 50 60 70 80 90 10 20 30 40 50 60 70 80 90 Rb1 M1 transformation rate (%) Effect of oral administration of Rb1 on Rb1(1 transformation rate# mice with low Rb1 M1 Parent Rb1-hydrolysing potential 10 25±11% Intestinal bacteria 8 Ginseng extract, Mean transformation rate 150 mg/kg x 14d (10 % <) Number of mice None 25± 11 % 6 Rb1 M1 Rb1-treated 51± 26 % transformation rate (%) 4 2 0 0- 10- 20- 30- 40- 50- 60- 70- 80- 90- 0- 10- 20- 30- 40- 50- 60- 70- 80- 90- 10 20 30 40 50 60 70 80 90 100 10 20 30 40 50 60 70 80 90 100 Rb1 M1 transformation rate (%)
  5. 5. Relationship between anti-metastatic potential and Rb1 metabolic activity## LLC Lung metastasis day 21day 0 Rb1, 25 mg/kg, p.o.Rb1 metabolic activity 60 50 Anti-metastatic activity Rb1(1ransformation rate M1 40 (% Inhibition) M1 M1 mice 30 Rb1-hydrolysing potential M1 with high hydrolyzing 46.8 ± 10.3 % M1 potential 20 M1 M1 10 M1 0 Rb1-hydrolysing potential mice 4.8 ± 3.2 % with low hydrolyzing M1 -10 potential 0 10 20 30 40 50 60 70 Rb1 M1 transformation rate (%) Effect of ginsenosides and their metabolites (M1, M4) on thegrowth of B16-BL6 melanoma, HT-1080 fibrosarcoma, and MLF fibroblastic cells in vitro
  6. 6. Effect of ginsenosides or their metabolites (M1, M4) on theinvasion of B16-BL6 melanoma and HT-1080 fibrosarcoma cells into Matrigel B16-BL6 melanoma Adhesion to 24 well subendothelial matrix cluster plate HT-1080 fibrosarcoma Enzymatic degradation of extracellular matrix Tumor cell Matrigel Microporous membrane Tumor cell migration (8 µm pore size) Laminin or Fibronectin 0.1%BSA in Medium (Lower compartment)Morphological changes of B16-BL6 melanoma and Lewis lung carcinoma (LLC) cells treated with ginsenoside-Rb1 and its metabolite M1 M1 (20 "M) Control Control M1 (40 "M) LLC cells Rb1 (40 "M) B16-BL6 cells
  7. 7. M1-induced DNA fragmentation of LLC cells and the cell morphology Control M1 (40 "M)Caspase-3 activity in LLC cells treated with M1 for various time periods ***, p < 0.001 activated-caspase-3 DEVD pNA DEVD pNA
  8. 8. Effect of caspase-3 inhibitor on the M1-induced Growth inhibition of LLC cells 120 100 ** ** % of control ** 80 * 60 40 20 0 Control 0 5 10 20 40 Pretreatment with caspase-3 inhibitor: Z-DEVD-FMK (µM) before the addition of 30 µM of M1Effect of M1 on the expression of caspase-3 mRNA in LLC cells
  9. 9. Cell cycle regulation c-Myc Phosphorylation Transcriptional activation Inhibition Inhibition InhibitionWestern blot analysis of p27Kip1, c-Myc and cyclin D1 in B16-BL6 cells treated with M1
  10. 10. Cell cycle regulation M1 c-Myc Phosphorylation Transcriptional activation Inhibition Inhibition InhibitionIntracellular distribution of M1 after the incubation of tumor cells with fluorescent-lablled dansyl M1 TLC profile of dansyl M1 and Fluorescent microscopy of its parent compound B16-BL6 cells treated with dansyl M1
  11. 11. Proposed mechanism of M1-induced inhibition of tumor growth Distribution of M1 after intravenous or oral administration to mice i.v. p.o.
  12. 12. TLC profiles of M1-metabolites in the liver after administration of M1 i.v. administration i.v. administration oral administration of M1 of M1 of M1 M1 0 10 20 40 80 160 min M1 Liver Contents M1 – + Livers after of small Livers administration intestine after (at 40 min) administration ( at 2 h) Putative metabolic pathways of ginsenosides in the body after oral administration EM1 M1 M1 M1 EM4 M4 M4

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