Man-Sau Wong

Inhibitory actions of genistein in human breast cancer (MCF-7) cells

Genistein, a natural isoflavanoid phytoestrogen, is thought to be the active ingredient in soy that possesses breast cancer preventive properties. The molecular mechanisms that are involved in its cancer preventive properties have not been completely understood. The present study is designed to investigate the mechanism involved in the inhibitory action of genistein in MCF-7 cells. Genistein at 50 and 100 microM significantly arrested the growth of MCF-7 cells at G2/M phase (P<0.05) and decreased at the proliferative S phase (P<0.05). Using cDNA microarray technology, genes differentially regulated by genistein were identified. In particular, as confirmed by reverse transcriptase-polymerase chain reaction (RT-PCR), genistein up-regulated heat shock protein 105 (HSP) mRNA and down-regulated mRNA expression of serum response factor (SRF), estrogen receptor (ER) alpha, disabled homolog 2 (DOC 2) and recombination activation gene 1 (RAG-1). Using real time RT-PCR, we have shown that HSP and SRF mRNA were both regulated by genistein in a time- and dose-dependent manner; however, it appears that only the effect of genistein on SRF mRNA, but not HSP mRNA expression, can be partially abolished by cotreatment with estrogen antagonist ICI 182,780. Western blotting analysis showed that the expressions of the ERalpha and SRF protein decreased significantly with genistein treatment (P<0.05). These results suggest that the inhibitory action of genistein on human breast cancer cells appears to be complex and is only partially mediated by the alteration of estrogen receptor-dependent pathways.

The osteoprotective effect of Herba epimedii (HEP) extract in vivo and in vitro

Herba epimedii (HEP) is one of the most frequently used herbs prescribed for treatment of osteoporosis in China. In the present study, the in vivo effects of HEP extract on bone metabolism were evaluated using 4-month-old ovariectomized (OVX) or sham-operated (Sham) female Sprague-Dawley rats orally administered with HEP extract (110 mg kg−1d−1), 17ß-estrogen (2 mg kg−1d−1) or its vehicle for 3 months. HEP extract significantly decreased urinary calcium excretion, suppressed serum alkaline phosphatase (ALP) activity and urinary deoxypyridinoline levels in OVX rats (P < 0.05 versus vehicle-treated OVX rats). Histomorphometric analysis indicated that HEP extract could prevent OVX-induced bone loss by increasing tibial trabecular bone area and decreasing trabecular separation in OVX rats (P < 0.05 versus vehicle-treated OVX group). The in vitro effects of HEP extract were also studied using rat osteoblast-like UMR 106 cells. HEP extract significantly stimulated cell proliferation in a dose-dependent manner (P < 0.01 versus vehicle-treated) and increased ALP activity at 200 μgml−1 (P < 0.01 versus vehicle-treated) in UMR 106 cells. It modulated osteoclastogenesis by increasing osteoprotegrin (OPG) mRNA and decreasing receptor activator of NF-κB ligand (RANKL) mRNA expression, resulting in a dose-dependent increase in OPG/RANKL mRNA ratio (P < 0.01 versus vehicle-treated). Taken together, HEP treatment can effectively suppress the OVX-induced increase in bone turnover possibly by both an increase in osteoblastic activities and a decrease in osteoclastogenesis. The present study provides the evidence that HEP can be considered as a complementary and alternative medicine for treatment of post-menopausal osteoporosis.

Icariin protects against bone loss induced by oestrogen deficiency and activates oestrogen receptor-dependent osteoblastic functions in UMR 106 cells

Background and purpose:  Icariin may be the active ingredient in Herba Epimedii, a Chinese herb commonly used for treatment of osteoporosis. The present study aims to delineate the mechanism(s) by which icariin prevents bone loss after ovariectomy (OVX) in vivo and stimulates osteoblastic functions in vitro.

Neuroprotective effects of genistein on dopaminergic neurons in the mice model of Parkinson's disease

Emerging evidence suggests beneficial effects of estrogen and estrogen-like chemicals on neurodegenerative diseases, especially Parkinsons disease (PD). Genistein, an isoflavone naturally found in soy products, displays estrogenic properties. The present study aims to investigate the neuroprotective effects of genistein on dopaminergic neurons in ovariectomized (OVX), 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-induced PD model mice. MPTP significantly decreased the levels of dopamine (DA) and its metabolites dihydroxyphenylacetic acid (DOPAC) and homovanillic acid (HVA) in the striatum, which could be restored by genistein or estrogen pretreatment. MPTP-challenge with genistein or estrogen pretreatment demonstrated reduced neurotoxicity, with tyrosine hydroxylase-immunoreactive (TH-IR) neurons in the substantia nigra pars compacta (SNpc) affected to a significantly lesser extent as compared to the MPTP treated control. The reverse transcription-PCR results also confirmed that the MPTP-induced downregulation of TH, dopamine transporter (DAT) and Bcl-2 mRNA expression in the midbrain could be restored by genistein or estrogen pretreatment. These findings provide the first evidence that genistein has neuroprotective effects on dopaminergic neurons in the MPTP-induced PD mice and this effect may be attributed to enhancing Bcl-2 gene expression.

Ginsenoside Rg1 protects dopaminergic neurons in a rat model of Parkinson's disease through the IGF-I receptor signalling pathway.

Background and purpose:  We have shown that ginsenoside Rg1 is a novel class of potent phytoestrogen and activates insulin‐like growth factor‐I receptor (IGF‐IR) signalling pathway in human breast cancer MCF‐7 cells. The present study tested the hypothesis that the neuroprotective actions of Rg1 involved activation of the IGF‐IR signalling pathway in a rat model of Parkinsons disease, induced by 6‐hydroxydopamine (6‐OHDA).

Naringin improves bone properties in ovariectomized mice and exerts oestrogen-like activities in rat osteoblast-like (UMR-106) cells.

Background and purpose:  Naringin, a flavanone glycoside in citrus fruits, has been recently reported to stimulate bone formation in vitro and in vivo. The present study was designed to determine if naringin could exert oestrogen‐like protective actions in bone.

BIOSENSOR MEASUREMENT OF THE INTERACTION KINETICS BETWEEN INSULIN-LIKE GROWTH FACTORS AND THEIR BINDING PROTEINS

The binding kinetics of human insulin-like growth factor binding protein (IGFBP) 1-6 for recombinant human insulin-like growth factor (IGF) I and II were measured and compared in the present study using surface plasmon resonance biosensor technique. Different concentrations of IGFBPs (5-100 nM) were allowed to interact with the immobilized IGF-I or IGF-II on sensor chip surface. Both des(1-3)IGF-I and insulin are known to bind weakly to the IGFBPs and therefore are used as negative controls for the binding experiments. The resultant sensorgrams were analyzed by using simple 1:1 binding model to derive both the association rate (k(a)) and dissociation rate (k(d)) constants for IGFBP-IGF interactions. The k(a) values of IGFBPs are in the range of 1x10(4) to 9x10(5) M(-1) s(-1) for IGF-I and 7x10(3) to 1.7x10(6) M(-1) s(-1) for IGF-II, respectively. The orders of k(a) for both IGF-I and IGF-II are IGFBP-3>IGFBP-5>IGFBP-6>IGFBP-4>IGFBP-2>++ +IGFBP-1. The k(d) values of IGFBPs are in the range of 1.5x10(-5) to 2x10(-4) s(-1) for IGF-I and 3.6x10(-5) to 3.7x10(-4) s(-1) for IGF-II, respectively. The order of k(d) for IGF-I is IGFBP-6>IGFBP-5>IGFBP-4>IGFBP-3>IGFBP-2>++ +IGFBP-1 and that for IGF-II is IGFBP-5>IGFBP-6>IGFBP-2>IGFBP-4>IGFBP-3>++ +IGFBP-1, respectively. The equilibrium affinity constants (K(A)) were calculated based on the ratio of k(a)/k(d) and were more precise than the published literature values based on competitive radioligand binding assays. The systematic study enables a direct comparison on the IGF-binding properties among the various IGFBPs, and the kinetic data provide additional information to delineate the physiological role of different IGFBPs in vivo.

Ginsenoside Rg1 exerts estrogen-like activities via ligand-independent activation of ERα pathway

Ginsenoside Rg1, an active ingredient commonly found in ginseng root, was previously demonstrated to be a phytoestrogen that exerted estrogen-like activity without direct interaction with estrogen receptors (ERs) in human breast cancer (MCF-7) cells. The present study was designed to determine the molecular mechanism by which Rg1 exerted estrogenic effects. Co-incubation of MCF-7 cells with 1 microM of ER antagonist ICI182780 abolished the inductive effects of Rg1 on pS2 expression as well as ERE-luciferase activity, suggesting that the estrogenic effects of Rg1 were mediated through the endogenous ERs. To evaluate the relative involvement of ERalpha and ERbeta in mediating the actions of Rg1, ER-negative human embryonic kidney (HEK293) cells were co-transfected with the ERE-luciferase reporter construct and either ERalpha or ERbeta construct. The results showed that Rg1 could activate ERE-luciferase activity via the ERalpha-mediated pathway in a dose-dependent manner (10(-14) to 10(-6)M); whereas, the activation of ERbeta-mediated ERE-luciferase activity by Rg1 only occur at high concentration (10(-6)M). Furthermore, the results showed that 1pM Rg1 could rapidly induce phosphorylation of the AF-1 domain of ERalpha at serine 118 residue within the first 5 min of incubation, suggesting that Rg1 activates ERalpha in a ligand-independent manner. Taken together, our results indicate that Rg1 preferentially activates ERalpha via phosphorylation of AF-1 domain in the absence of receptor binding. This study is the first to provide evidence that ginsenoside Rg1 exerts estrogen-like actions via ligand-independent activation of ERalpha pathway.

Molecular mechanisms of survival and apoptosis in RAW 264.7 macrophages under oxidative stress

Organisms living in an aerobic environment are continuously exposed to reactive oxygen species (ROS). Apoptosis of cells can be induced by ROS and cells also develop negative feedback mechanisms to limit ROS induced cell death. In this study, RAW264.7 murine macrophage cells were treated with H2O2 and cDNA microarray technique was used to produce gene expression profiles. We found that H2O2 treatment caused up-regulation of stress, survival and apoptosis related genes, and down-regulation of growth and cell cycle promoting genes. Numerous genes of metabolism pathways showed special expression patterns under oxidative stress: glycolysis and lipid synthesis related genes were down-regulated whereas the genes of lipid catabolism and protein synthesis were up-regulated. We also identified several signaling molecules as ROS-responsive, including p53, Akt, NF-κ B, ERK, JNK, p38, PKC and INF-γ . They played important roles in the process of apoptosis or cell survival. Finally, an interactive pathway involved in cellular response to oxidative stress was proposed to provide some insight into the molecular events of apoptosis induced by ROS and the feedback mechanisms involved in cell survival.

Proteome of Oriental ginseng Panax ginseng C. A. Meyer and the potential to use it as an identification tool

Oriental ginseng (Panax ginseng C. A. Meyer) and American ginseng (Panax quinquefolius) are two widely used valuable traditional Chinese medicines (TCM). Previously, the identification of ginseng was mainly performed by analyzing the ginsengnosides using high performance liquid chromatography and amplification of polymorphic DNA using polymerase chain reaction. However, these methods cannot be used to distinguish TCM samples which are from different parts (main root, lateral roots, rhizome head and skin) of ginseng and ginseng culture cells from wild‐grown ginseng. The present study aimed to identify different species of ginseng, different parts of the same ginseng and cultured cells of ginseng using a proteomic approach. Two‐dimensional electrophoresis (2‐DE) maps were established from the American ginseng main root, different parts (main root, lateral roots, rhizome head and skins) of Oriental ginseng and Oriental ginseng culture cells. Our results show that the 2‐DE maps of different ginseng samples contain sufficient differences to permit easy discrimination. We have also identified common and specific protein spots in the 2‐DE maps of different ginseng samples. The use of these “marker proteins” may help to speed up the identification process.