Jin-Rong Zhou

Soy Phytochemicals Prevent Orthotopic Growth and Metastasis of Bladder Cancer in Mice by Alterations of Cancer Cell Proliferation and Apoptosis and Tumor Angiogenesis

A role of dietary bioactive components in bladder cancer prevention is biologically plausible because most substances or metabolites are excreted through the urinary tract and are consequently in direct contact with the mucosa of the bladder. We first determined antigrowth activity of genistein against poorly differentiated 253J B-V human bladder cancer cells in vitro. Genistein inhibited the cell growth in a time- and dose-dependent manner via G(2)-M arrest, down-regulation of nuclear factor kappaB (NF-kappaB), and induction of apoptosis. We also evaluated both genistin, which is a natural form of genistein, and the isoflavone-rich soy phytochemical concentrate (SPC) on the growth and metastasis of 253J B-V tumors in an orthotopic tumor model. Mice treated with genistin and SPC had reduced final tumor weights by 56% (P < 0.05) and 52% (P < 0.05), respectively, associated with induction of tumor cell apoptosis and inhibition of tumor angiogenesis in vivo. In addition, SPC treatment, but not genistin treatment, significantly inhibited lung metastases by 95% (P < 0.01) associated with significant down-regulation of NF-kappaB expression in tumor tissues and reduction of circulating insulin-like growth factor-I levels, suggesting that SPC may contain other bioactive ingredients that have antimetastatic activity. The results from our studies suggest that further clinical investigation should be warranted to apply soy phytochemicals, such as SPC, as a potent prevention regimen for bladder cancer progression. This orthotopic human bladder tumor model also provides a clinically relevant experimental tool for assessing potential preventive activity of other dietary components against bladder tumor growth and metastasis.

COMBINED INHIBITION OF ESTROGEN-DEPENDENT HUMAN BREAST CARCINOMA BY SOY AND TEA BIOACTIVE COMPONENTS IN MICE

Breast cancer is significantly less prevalent among Asian women, whose diets contain high intake of soy products and tea. The objective of our present study was to identify the combined effects of dietary soy phytochemicals and tea components on breast tumor progression in a clinically relevant in vivo model of MCF‐7 androgen‐dependent human breast tumor in female SCID mice. MCF‐7 tumor growth, tumor cell proliferation and apoptosis, microvessel density, and expressions of tumor estrogen receptors were compared in mice treated with genistin‐rich soy isoflavones (GSI), soy phytochemical concentrate (SPC), black tea (BT), green tea (GT), SPC/BT combination and SPC/GT combination. GSI and SPC led to dose‐dependent inhibition of MCF‐7 tumor growth via inhibition of cancer cell proliferation in vivo. GT showed more potent anti‐breast tumor activity than BT. GT infusion at 1.5 g tealeaf/100 mL water produced significant (p < 0.05) reductions of 56% in final tumor weight. GT plus SPC at 0.1% of the diet further reduced final tumor weight by 72% (p < 0.005). Analysis of serum and tumor biomarkers showed that the combined effects of SPC and GT inhibited tumor angiogenesis, and reduced estrogen receptor (ER)‐α and serum levels of insulin‐like growth factor (IGF)‐I. Our study suggests that dietary SPC plus GT may be used as a potential effective dietary regimen for inhibiting progression of estrogen‐dependent breast cancer.

Bioactive tanshinones in Salvia miltiorrhiza inhibit the growth of prostate cancer cells in vitro and in mice

Searching for efficacious and safe agents for the chemoprevention and therapy of prostate cancer has become the top priority of research. The objective of this study was to determine the effects of a group of tanshinones from a Chinese herb Salvia Miltiorrhiza, cryptotanshinone (CT), tanshinone IIA (T2A) and tanshinone I (T1) on prostate cancer. The in vitro studies showed that these tanshinones inhibited the growth of human prostate cancer cell lines in a dose‐dependent manner via cell cycle arrest and apoptosis induction. Among three compounds, T1 had the most potent activity with IC50s around 3–6 μM. On the other hand, tanshinones had much less adverse effects on the growth of normal prostate epithelial cells. The epigenetic pathway focused array assay identified Aurora A kinase as a possible target of tanshinone actions. The expression of Aurora A was overexpressed in prostate cancer cell lines. Moreover, knockdown of Aurora A in prostate cancer cells significantly decreased cell growth. Tanshinones significantly downregulated the Aurora A expression, suggesting Aurora A may be a functional target of tanshinones. Tanshinones, especially T1, also showed potent anti‐angiogenesis activity in vitro and in vivo. Furthermore, T1 inhibited the growth of DU145 prostate tumor in mice associated with induction of apoptosis, decrease of proliferation, inhibition of angiogenesis and downregulation of Aurora A, whereas it did not alter food intake or body weight. Our results support that T1 may be an efficacious and safe chemopreventive or therapeutic agent against prostate cancer progression.

Daidzein-rich isoflavone aglycones are potentially effective in reducing hot flashes in menopausal women.

Objective: The aim of this study was to determine the effect of DRIs on hot flash symptoms in menopausal women. Design: This was a randomized, double-blind, placebo-controlled trial of menopausal women, aged 38 to 60 years, who experienced 4 to 14 hot flashes per day. After a 1-week run-in period, a total of 190 menopausal women were randomized to receive a placebo or 40 or 60 mg/day of a DRI for 12 weeks. The primary outcome was the mean changes from baseline to week 12 in the frequency of hot flashes recorded in the participant diary. The secondary outcomes included changes in quality of life and hormonal profiles. Results: A total of 147 women (77%) completed the study. It was found that 40 and 60 mg of DRI improved hot flash frequency and severity equally. At 8 weeks hot flash frequency was reduced by 43% in the 40-mg DRI group and by 41% in the 60-mg DRI group, compared with 32% in the placebo group (P = not significant vs placebo). The corresponding numbers for 12 weeks were 52%, 51%, and 39%, respectively (P = 0.07 and 0.09 vs placebo). When comparing the two treatment groups with the placebo group, there were significant reductions in mean daily hot flash frequency. The supplement (either 40 or 60 mg) reduced hot flash frequency by 43% at 8 weeks (P = 0.1) and 52% at 12 weeks (P = 0.048) but did not cause any significant changes in endogenous sex hormones or thyroid hormones. Menopausal quality of life improved in all three groups, although there were no statistically significant differences between groups. Conclusions: DRI supplementation may be an effective and acceptable alternative to hormone treatment for menopausal hot flashes.

Genistein sensitizes inhibitory effect of tamoxifen on the growth of estrogen receptor-positive and HER2-overexpressing human breast cancer cells

Although tamoxifen (TAM) is used for the front‐line treatment and prevention of estrogen receptor‐positive (ER+) breast tumors, nearly 40% of estrogen‐dependent breast tumors do not respond to TAM treatment. Moreover, the positive response is usually of short duration, and most tumors eventually develop TAM‐resistance. Overexpression of HER2 gene is associated with TAM‐resistance of breast tumor, and suppression of HER2 expression enhances the TAM activity. Soy isoflavone genistein has been shown to have anti‐cancer activities and suppress expression of HER2 and ERα. The objective of this study was to test the hypothesis that genistein may sensitize the response of ER+ and HER2‐overexpressing breast cancer cells to TAM treatment. The combination treatment of TAM and genistein inhibited the growth of ER+/HER2‐overexpressing BT‐474 human breast cancer cells in a synergistic manner in vitro. Determination of cellular markers indicated that this synergistic inhibitory effect might be contributed in part from combined effects on cell‐cycle arrest at G1 phase and on induction of apoptosis. Further determination of the molecular markers showed that TAM and genistein combination synergistically induced BT‐474 cell apoptosis in part by synergistic downregulation of the expression of survivin, one of the apoptotic effectors, and downregulation of EGFR, HER2, and ERα expression. Our research may provide a novel approach for the prevention and/or treatment of TAM insensitive/resistant human breast cancer, and warrants further in vivo studies to verify the efficacy of genistein and TAM combination on the growth of ER+/HER2‐overexpressing breast tumors and to elucidate the in vivo mechanisms of synergistic actions.

Effect of Soy Nuts on Adhesion Molecules and Markers of Inflammation in Hypertensive and Normotensive Postmenopausal Women

Recently, it was shown that substituting soy nuts for nonsoy protein in a therapeutic lifestyle change (TLC) diet lowered systolic and diastolic blood pressure by 9.9% and 6.8%, respectively, in postmenopausal women with hypertension and by 5.2% and 2.9%, respectively, in normotensive postmenopausal women. In this study, to examine mechanisms for these reductions, markers of inflammation were measured, including soluble vascular cell adhesion molecule-1, soluble intercellular adhesion molecule-1, C-reactive protein, interleukin-6, and matrix metalloproteinase-9. Sixty healthy postmenopausal women (48 normotensive and 12 with hypertension) were randomized in a crossover design to a TLC diet alone or a TLC diet in which 0.5 cups of soy nuts (25 g soy protein and 101 mg aglycone isoflavones) replaced 25 g of nonsoy protein daily. Each diet was followed for 8 weeks. Compared with the TLC diet alone, levels of soluble vascular cell adhesion molecule-1 were significantly lower on the soy diet in women with hypertension (623.6 +/- 153.8 vs 553.8 +/- 114.4 ng/ml, respectively, p = 0.003), whereas no significant differences were observed in normotensive women. Soy nuts were associated with a trend toward reduction in C-reactive protein in normotensive women. No effect on levels of soluble intercellular adhesion molecule-1, interleukin-6, or matrix metalloproteinase-9 was observed. In conclusion, the reduction in soluble vascular cell adhesion molecule-1 with soy nuts in women with hypertension suggests an improvement in endothelial function that may reflect an overall improvement in the underlying inflammatory process underlying atherosclerosis.

Epigenetic Alterations of the Dopaminergic System in Major Psychiatric Disorders

Although there is evidence to link schizophrenia (SCZ) and bipolar disorder (BD) to genetic and environmental factors, specific individual or groups of genes/factors causative of the disease have been elusive to the research community. An understanding of the molecular aberrations that cause these mental illnesses requires comprehensive approaches that examine both genetic and epigenetic factors. Because of the overwhelming evidence for the role of environmental factors in the disease presentation, our initial approach involved deciphering how epigenetic changes resulting from promoter DNA methylation affect gene expression in SCZ and BD. Apparently, the central reversible but covalent epigenetic modification to DNA is derived from methylation of the cytosine residues that is potentially heritable and can affect gene expression and downstream activities. Environmental factors can influence DNA methylation patterns and hence alter gene expression. Such changes can be especially problematic in individuals with genetic susceptibilities to specific diseases. Recent reports from our laboratory provided compelling evidence that both hyper- and hypo-DNA methylation changes of the regulatory regions play critical roles in defining the altered functionality of genes in major psychiatric disorders such as SCZ and BD. In this chapter, we outline the technical details of the methods that could help to expand this line of research to assist with compiling the differential methylation-mediated epigenetic alterations that are responsible for the pathogenesis of SCZ, BD, and other mental diseases. We use the genes of the extended dopaminergic (DAergic) system such as membrane-bound catechol-O-methyltransferase (MB-COMT), monoamine oxidase A (MAOA), dopamine transporter 1 (DAT1), tyrosine hydroxylase (TH), dopamine (DA) receptors1 and 2 (DRD1/2), and related genes (e.g., reelin [RELN] and brain-derived neurotrophic factor [BDNF]) to illustrate the associations between differential promoter DNA methylations and disease phenotype. It is our hope that comprehensive analyses of the DAergic system as the prototype could provide the impetus and molecular basis to uncover early markers for diagnosis, help in the understanding of differences in disease severity in individuals with similar or identical genetic makeup, and assist with the identification of novel targets for therapeutic applications.

A novel pathway involving melanoma differentiation associated gene-7/interleukin-24 mediates nonsteroidal anti-inflammatory drug-induced apoptosis and growth arrest of cancer cells.

Numerous studies show that nonsteroidal anti-inflammatory drugs (NSAIDs) are effective in chemoprevention or treatment of cancer. Nevertheless, the mechanisms underlying these antineoplastic effects remain poorly understood. Here, we report that induction of the cancer-specific proapoptotic cytokine melanoma differentiation associated gene-7/interleukin-24 (MDA-7/IL-24) by several NSAIDs is an essential step for induction of apoptosis and G(2)-M growth arrest in cancer cells in vitro and inhibition of tumor growth in vivo. We also show that MDA-7/IL-24-dependent up-regulation of growth arrest and DNA damage inducible 45 alpha (GADD45alpha) and GADD45gamma gene expression is sufficient for cancer cell apoptosis via c-Jun NH(2)-terminal kinase (JNK) activation and growth arrest induction through inhibition of Cdc2-cyclin B checkpoint kinase. Knockdown of GADD45alpha and GADD45gamma transcription by small interfering RNA abrogates apoptosis and growth arrest induction by the NSAID treatment, blocks JNK activation, and restores Cdc2-cyclin B kinase activity. Our results establish MDA-7/IL-24 and GADD45alpha and GADD45gamma as critical mediators of apoptosis and growth arrest in response to NSAIDs in cancer cells.

Progression to androgen‐independent LNCaP human prostate tumors: Cellular and molecular alterations

Lethal phenotypes of human prostate cancer are characterized by progression to androgen‐independence and metastasis. For want of a clinically relevant animal model, mechanisms behind this progression remain unclear. Our study used an in vivo model of androgen‐sensitive LNCaP human prostate cancer cell xenografts in male SCID mice to study the cellular and molecular biology of tumor progression. Primary tumors were established orthotopically, and the mice were then surgically castrated to withdraw androgens. Five generations of androgen‐independent tumors were developed using castrated host mice. Tumor samples were used to determine expressions of cellular and molecular markers. Androgen‐independent tumors had increased proliferation and decreased apoptosis compared to androgen‐sensitive tumors, outcomes associated with elevated expression of p53, p21/waf1, bcl‐2, bax and the bcl‐2/bax ratio. Blood vessel growth in androgen‐independent tumor was associated with increased expression of vascular endothelial growth factor. Overexpression of androgen receptor mRNA and reduced expression of androgen receptor protein in androgen‐independent tumors suggest that the androgen receptor signaling pathway may play an important role in the progression of human prostate cancer to androgen‐independence. The in vivo orthotopic LNCaP tumor model described in our study mimics the clinical course of human prostate cancer progression. As such, it can be used as a model for defining the molecular mechanisms of prostate cancer progression to androgen‐independence and for evaluating the effect of preventive or therapeutic regimens for androgen‐independent human prostate cancer.

Tanshinones Inhibit the Growth of Breast Cancer Cells through Epigenetic Modification of Aurora A Expression and Function

The objectives of this study were to evaluate the effects of tanshinones from a Chinese herb Salvia Miltiorrhiza on the growth of breast cancer cells, and to elucidate cellular and molecular mechanisms of action. Tanshinones showed the dose-dependent effect on the growth inhibition of breast cancer cells in vitro, with tanshinone I (T1) the most potent agent. T1 was also the only tanshinone to have potent activity in inhibiting the growth of the triple-negative breast cancer cell line MDA-MB231. T1 caused cell cycle arrests of both estrogen-dependent and estrogen-independent cell lines associated with alterations of cyclinD, CDK4 and cyclinB, and induced breast cancer cell apoptosis associated with upregulation of c-PARP and downregulation of survivin and Aurora A. Among these associated biomarkers, Aurora A showed the most consistent pattern with the anti-growth activity of tanshinones. Overexpression of Aurora A was also verified in breast tumors. The gene function assay showed that knockdown of Aurora A by siRNA dramatically reduced the growth-inhibition and apoptosis-induction activities of T1, suggesting Aurora A as an important functional target of T1 action. On the other hand, tanshinones had much less adverse effects on normal mammary epithelial cells. Epigenetic mechanism studies showed that overexpression of Aurora A gene in breast cancer cells was not regulated by gene promoter DNA methylation, but by histone acetylation. T1 treatment significantly reduced acetylation levels of histone H3 associated with Aurora A gene. Our results supported the potent activity of T1 in inhibiting the growth of breast cancer cells in vitro in part by downregulation of Aurora A gene function. Our previous studies also demonstrated that T1 had potent anti-angiogenesis activity and minimal side effects in vivo. Altogether, this study warrants further investigation to develop T1 as an effective and safe agent for the therapy and prevention of breast cancer.