Characterization of the molecular action of ginsenoside Rg1

Pao Yue-kong Library Electronic Theses Database

Characterization of the molecular action of ginsenoside Rg1


Author: Lau, Wai-sum
Title: Characterization of the molecular action of ginsenoside Rg1
Degree: M.Phil.
Year: 2006
Subject: Hong Kong Polytechnic University -- Dissertations
Ginseng -- Therapeutic use
Department: Dept. of Applied Biology and Chemical Technology
Pages: xxiii, 162 leaves : ill. ; 30 cm
Language: English
InnoPac Record:
Abstract: Ginsenoside Rgl, an active ingredient commonly found in ginseng root, was previously demonstrated to be a novel class of phytoestrogens. It can stimulate the growth of human breast cancer (MCF-7) cells as well as activate the estrogen response element (ERE)-dependent luciferase activities in HeLa cells. Interestingly, the action of Rgl is estrogen receptor alpha (ERa)-dependent but it does not interact directly with this receptor. Thus, we hypothesize that Rgl may mediate its estrogen-like actions in MCF-7 cell via ligand-independent activation of ER pathway. It is well documented that ER cross-talk with insulin-like growth factor-I receptor (IGF-IR) mediated pathway. Therefore, the present study aims to characterize the molecular action of Rgl in the IGF-IR mediated pathway and to determine if tyrosine kinase and the downstream signaling molecule mitogen-activated protein kinase kinase (MEK) are involved. In order to investigate the role of tyrosine kinase in the action of Rgl, MCF-7 cells treated with 1pM Rgl were co-incubated with 50uM genistein (tyrosine kinase inhibitor) for 48h. Their effects on cell proliferation rate, estrogen response element-luciferase reporter gene activities and the protein expression of IGF-IR, src-homology-collagen (She) and ERa were then measured. Our results showed that Rgl could increase the cell proliferation rate, luciferase activities and expression of IGF-IR protein significantly p<0.05) in MCF-7 cells. When MCF-7 cells were cotreated with 50uM genistein, the proliferative effects of Rgl and its induction of IGF-IR protein expression were completely abolished. These results showed that the proliferative effect of Rgl in MCF-7 cells is at least in part mediated by the induction of IGF-I receptor expression and its action involved tyrosine kinase-dependent pathways. Similarly, 50uM PD98059 (a specific MEK inhibitor) was used to investigate the role of MEK in the action of Rgl. After treatment of MCF-7 cells for 48h, all the inductive effects of Rgl on cell proliferation, luciferase activity and expression of IGF-1R protein were abolished by co-treatment with 50uM PD98059. These results showed that estrogen-like actions of Rgl in MCF-7 cells are MEK- dependent. To determine the sequence of activation of MEK and ERa by Rgl in MCF-7 cells, the expression and activation of MEK and ERa in Rgl-treated MCF-7 cells were studied. The protein expression and phosphorylation levels of MEK and ERa in MCF-7 cells in response to treatment with Rgl for different period of time were studied. The results of western blotting showed that the activation of MEK by 1 pM Rgl began at 5 min, and by 48 h, the phosphorylation and protein expression of MEK were significantly upregulated (p<0.05). Moreover, we found that treatment of MCF-7 cells with 1pM of Rgl for 5 min could phosphorylate the Ser118 site of ERa significantly (p<0.05) without altering its protein expression. As our results indicated that tyrosine kinase was involved in the action of Rgl, we then investigated if the activation of tyrosine kinase involved direct binding between IGF-IR and Rgl. MCF-7 cells treated with 1pM Rgl were co-incubated with 10uM H1356 (IGF-IR antagonist that bind to extracellular domain of IGF-IR). The phosphorylation of MEK was measured after treatment of MCF-7 cells for 10 min. The result showed that the Rgl -mediated phosphorylaton of MEK could not be inhibited by H1356. This data indicated that Rgl might not interact directly with the extracellular domain of IGF-IR for activation of tyrosine kinase. The molecular action of Rgl was also studied using an ER-negative human embryonic kidney cell (HEK293). The abilities of Rgl to activate ERa or ERb-dependent ERE-luciferase constructs were examined by using transient transfection of HEK293 with the corresponding plasmids. The results of luciferase assay showed that Rgl could stimulate ERa stronger than that of ERb and the activations were in a concentration-dependent manner. The results indicated that Rgl can activate both isoforms of ER in transfected HEK cells. We then determined if Rgl also activate MEK in ER-negative HEK293 cells. MEK phosphorylation in ER-negative HEK293 cells increased upon treatment with 1pM Rgl for 10 minutes. Such activation could be completely abolished by co-treatment of HEK293 cells with 50uM genistein (tyrosine kinase inhibitor). The results indicated that the activation of MEK by Rgl in HEK293 cells is at least in part mediated by the action of tyrosine kinase and such activation does not require the presence of ERs. In conclusion, these results showed that the proliferative effect of Rgl in MCF-7 cells involved the activation of ER and its action could be mediated by both tyrosine kinase- and MEK- dependent pathways. These data provide new evidence to support our hypothesis that Rgl activates ER in a ligand-independent manner in MCF-7 cells.

Files in this item

Files Size Format
b19579834.pdf 4.027Mb PDF
Copyright Undertaking
As a bona fide Library user, I declare that:
  1. I will abide by the rules and legal ordinances governing copyright regarding the use of the Database.
  2. I will use the Database for the purpose of my research or private study only and not for circulation or further reproduction or any other purpose.
  3. I agree to indemnify and hold the University harmless from and against any loss, damage, cost, liability or expenses arising from copyright infringement or unauthorized usage.
By downloading any item(s) listed above, you acknowledge that you have read and understood the copyright undertaking as stated above, and agree to be bound by all of its terms.


Quick Search


More Information