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
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
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
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
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
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
Proposed mechanism of M1-induced inhibition of tumor growth Distribution of M1 after intravenous or oral administration to mice i.v. p.o.
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