Wen-Fang Chen

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.

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

Background and purpose:u2002 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:u2002 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).

Ginsenoside Rg1 protects against 6-OHDA-induced neurotoxicity in neuroblastoma SK-N-SH cells via IGF-I receptor and estrogen receptor pathways

Our previous studies have demonstrated that ginsenoside Rg1 is a novel class of potent phytoestrogen and can mimic the action of estradiol in stimulation of MCF‐7 cell growth by the crosstalk between insulin‐like growth factor‐I receptor (IGF‐IR)‐dependent pathway and estrogen receptor (ER)‐dependent pathway. The present study was designed to investigate the neuroprotective effects of ginsenoside Rg1 against 6‐hydroxydopamine (6‐OHDA)‐induced neurotoxicity in human neuroblastoma SK‐N‐SH cells and the possible mechanisms. Pre‐treatment with ginsenoside Rg1 resulted in an enhancement of survival, and significant rescue occurred at the concentration of 0.01u2003μM on cell viability against 6‐OHDA‐induced neurotoxicity. These effects could be completely blocked by IGF‐IR antagonist JB‐1 or ER antagonist ICI 182780. 6‐OHDA arrested the cells at G0G1 phase and prevented S phase entry. Rg1 pre‐treatment could reverse the cytostatic effect of 6‐OHDA. Ginsenoside Rg1 also could attenuate 6‐OHDA‐induced decrease in mitochondrial membrane potential. These effects could also be completely blocked by JB‐1 or ICI 182780. Furthermore, 6‐OHDA‐induced up‐regulation of Bax and down‐regulation of Bcl‐2 mRNA and protein expression could be restored by Rg1 pre‐treatment. Rg1 pre‐treatment could reverse the down‐regulation of ERα protein expression induced by 6‐OHDA treatment. Cells transfected with the estrogen responsive element (ERE)‐luciferase reporter construct exhibited significantly increased ERE‐luciferase activity in the Rg1 presence, suggesting that the estrogenic effects of Rg1 were mediated through the endogenous ERs. These results suggest that ginsenoside Rg1 may attenuate 6‐OHDA‐induced apoptosis and its action might involve the activation of IGF‐IR signaling pathway and ER signaling pathway.

Mitogen‐activated protein kinase (MAPK) pathway mediates the oestrogen‐like activities of ginsenoside Rg1 in human breast cancer (MCF‐7) cells

Background and purpose:u2002 The present study was designed to determine how ginsenoside Rg1, an active ingredient in ginseng root, exerts its oestrogenic effects. We hypothesize that Rg1 may exert oestrogen‐like actions in MCF‐7 cells by activating the mitogen‐activated protein kinase (MAPK) pathway in a ligand‐independent manner.

Ginsenoside Rg1 protects against 6-OHDA-induced toxicity in MES23.5 cells via Akt and ERK signaling pathways.

AIM OF THE STUDYnThe present study was designed to investigate the neuroprotective effects of ginsenoside Rg1 against 6-hydroxydopamine (6-OHDA)-induced toxicity in MES23.5 cells and their possible mechanisms.nnnMATERIALS AND METHODSnMES23.5 cells were treated with or without Rg1 for 24h before exposure to 6-OHDA. Cell viability was determined by MTS assay. The gene and protein expressions of Bcl-2 were detected by real time RT-PCR and western blotting. Phosphorylation of Akt and ERK1/2 were examined by western blotting.nnnRESULTSnPretreatment with ginsenoside Rg1 had obvious neuroprotective effects on cell viability against 6-OHDA-induced toxicity. 6-OHDA decreased the gene and protein expressions of Bcl-2. These effects could be reversed by Rg1 pretreatment. Potential cell signaling candidates involved in this neuroprotective effect were examined. 6-OHDA significantly inhibited the phosphorylation of Akt and increased the phosphorylation of ERK1/2 in MES23.5 cells. Pretreatment with ginsenoside Rg1 could increase the Akt phosphorylation and inhibit the ERK1/2 phosphorylation induced by 6-OHDA. Further study revealed that LY294002, an inhibitor of phosphatidylinositol 3-kinase (PI3-K), attenuated the neuroprotective effect of Rg1 on cell viability against 6-OHDA-induced toxicity.nnnCONCLUSIONSnTaken together, our results strongly suggest that ginsenoside Rg1 has neuroprotective effects against 6-OHDA-induced toxicity in MES23.5 cells. Its mechanism includes the up-regulation of Bcl-2 gene expression, the activation of Akt phoshphorylation as well as the inhibition of ERK1/2 phosphorylation induced by 6-OHDA.

Activation of insulin-like growth factor I receptor-mediated pathway by ginsenoside Rg1

Ginsenoside Rg1, an active ingredient in ginseng, was previously shown to be a novel class of potent phytoestrogen. The present study aims at investigating the molecular mechanisms involved in mediating its actions in human breast cancer (MCF‐7) cells. Rg1 (1u2003pM) stimulates cell proliferation (P<0.01) and estrogen‐responsive pS2 mRNA expression (P<0.05) without alteration of estrogen receptor alpha (ERα) protein or mRNA expression in MCF‐7 cells. In addition, 10−14–10−4u2003M of Rg1 does not demonstrate specific binding to ERα. We hypothesize that Rg1 may exert its actions in MCF‐7 cell via the activation of crosstalk between ER‐ and insulin growth factor I receptor (IGF‐IR)‐dependent pathways. The results indicate that Rg1 significantly increases IGF‐IR expression and IGF‐IR promoter activity in MCF‐7 cells (P<0.05). Cotreatment of MCF‐7 cells with 1u2003μM of estrogen antagonist ICI 182,780 completely abolishes the effects of Rg1 on IGF‐IR expression. Furthermore, Rg1 enhances tyrosine phosphorylation of IRS‐1 in MCF‐7 cells upon IGF‐I stimulation and the activation of IRS‐1 phosphorylation is also ER‐dependent. Taken together, our results suggest that Rg1 not only increases IGF‐IR expression but also enhances IGF‐IR‐mediated signaling pathways in MCF‐7 cells. The stimulation of IGF‐IR expression by Rg1 in MCF‐7 cells appears to require ER, and its actions might involve ligand‐independent activation of ER.

Soy isoflavones and their bone protective effects

Abstract.Several observational studies have suggested that populations with a high dietary soy intake have a lower incidence of osteoporosis-related fractures when compared to Western populations. However, there has not been consistent data to show that soy isoflavones protect against or lessen bone loss. Studies in our laboratory showed that genistein, the major soy isoflavone, could stimulate osteoblastic functions as well as human breast cancer cell growth. These studies raised the concern of whether it would be safe for women who have a prior history of breast cancer to consume soy isoflavone for management of postmenopausal osteoporosis. As increasing the purity of genistein is known to increase its ability to induce human breast cancer cell growth, current effort in our laboratory is to determine if the in vivo bone protective effects will be affected by the complexity of the soy isoflavones extract in ovariectomized mice.

Total flavonoid fraction of the Herba epimedii extract suppresses urinary calcium excretion and improves bone properties in ovariectomised mice

Flavonoids are the active components of Herba epimedii (HEP), a commonly used herb for the management of osteoporosis in China over the centuries. The present study aims to characterise the in vivo effects of its total flavonoid (TF) fraction on bone properties and mineral metabolism as well as to study the mechanism involved in achieving its protective effects against ovariectomy (OVX)-induced bone loss. TF suppressed OVX-induced increase in urinary Ca excretion as well as loss of bone mass and strength at the distal femur in mice in a dose-dependent manner. The changes in urinary Ca excretion were inversely correlated with the expressions of renal Ca transport protein (CaBP-28K) and vitamin D receptor mRNA in OVX mice. TF (100 μg/g) treatment prevented the deterioration of trabecular bone microarchitecture induced by OVX in mice. In addition, TF treatment increased the expression of type I collagen and osteocalcin mRNA and the ratio of osteoprotegerin/receptor activator of NF-κB ligand mRNA, and suppressed the increase in IL-6 mRNA induced by OVX in the femur of mice. The present results indicate that the optimal dosage of the TF fraction of HEP for the improvement of bone properties and mineral metabolism in OVX mice was between 50 and 100 μg/g. Mechanistic studies indicated that TF might increase renal Ca reabsorption, stimulate the process of osteoblast formation as well as suppress the process of osteoclastogenesis in OVX mice.