Role for Nongenomic Estrogen Signaling in Male Fertility.

Estrogen actions are mediated by both nuclear (n) and membrane (m) localized estrogen receptor 1 (ESR1). Male Esr1 knockout (Esr1KO) mice lacking functional Esr1 are infertile, with reproductive tract abnormalities. Male mice expressing nESR1 but lacking mESR1 (nuclear-only estrogen receptor 1 mice) are progressively infertile due to testicular, rete testis, and efferent ductule abnormalities similar to Esr1KO males, indicating a role for mESR1 in male reproduction. The H2NES mouse expresses only mESR1 but lacks nESR1. The goal of this study was to identify the functions of mESR1 alone in mice where nESR1 was absent. Breeding trials showed that H2NES males are fertile, with decreased litter numbers but normal pup numbers/litter. In contrast to Esr1KO mice, H2NES testicular, and epididymal weights were not reduced, and seminiferous tubule abnormalities were less pronounced. However, Esr1KO and H2NES males both had decreased sperm motility and a high incidence of abnormal sperm morphology. Seminiferous tubule and rete testis dilation and decreased efferent ductule epithelial height characteristic of Esr1KO males were reduced in H2NES. Consistent with this, expression of genes involved in fluid transport and ion movement that were reduced in Esr1KO (Aqp1, Car2, Car14, Cftr) were partially or fully restored to wild-type levels in H2NES. In summary, in contrast to Esr1KO males, H2NES males are fertile and have reduced phenotypic and functional abnormalities in the testis and efferent ductules. Thus, mESR1 alone, in the absence of nESR1, can partially regulate male reproductive tract structure and function, emphasizing its importance for overall estrogen action.

Uterine-specific Ezh2 deletion enhances stromal cell senescence and impairs placentation, resulting in pregnancy loss.

Maternal uterine remodeling facilitates embryo implantation, stromal cell decidualization and placentation, and perturbation of these processes may cause pregnancy loss. Enhancer of zeste homolog 2 (EZH2) is a histone methyltransferase that epigenetically represses gene transcription; loss of uterine EZH2 affects endometrial physiology and induces infertility. We utilized a uterine Ezh2 conditional knockout (cKO) mouse to determine EZH2's role in pregnancy progression. Despite normal fertilization and implantation, embryo resorption occurred mid-gestation in Ezh2cKO mice, accompanied by compromised decidualization and placentation. Western blot analysis revealed Ezh2-deficient stromal cells have reduced amounts of the histone methylation mark H3K27me3, causing upregulation of senescence markers p21 and p16 and indicating that enhanced stromal cell senescence likely impairs decidualization. Placentas from Ezh2cKO dams on gestation day (GD) 12 show architectural defects, including mislocalization of spongiotrophoblasts and reduced vascularization. In summary, uterine Ezh2 loss impairs decidualization, increases decidual senescence, and alters trophoblast differentiation, leading to pregnancy loss.

Identification and characterization of novel abdominal and pelvic brown adipose depots in mice.

Brown adipose tissue (BAT) generates heat through non-shivering thermogenesis, and increasing BAT amounts or activity could facilitate obesity treatment and provide metabolic benefits. In mice, BAT has been reported in perirenal, thoracic and cranial sites. Here, we describe new pelvic and lower abdominal BAT depots located around the urethra, internal reproductive and urinary tract organs and major lower pelvic blood vessels, as well as between adjacent muscles where the upper hind leg meets the abdominal cavity. Immunohistochemical, western blot and PCR analyses revealed that these tissues expressed BAT markers such as uncoupling protein 1 (UCP1) and CIDEA, but not white adipose markers, and ß3-adrenergic stimulation increased UCP1 amounts, a classic characteristic of BAT tissue. The newly identified BAT stores contained extensive sympathetic innervation with high mitochondrial density and multilocular lipid droplets similar to interscapular BAT. BAT repositories were present and functional neonatally, and showed developmental changes between the neonatal and adult periods. In summary, several new depots showing classical BAT characteristics are reported and characterized in the lower abdominal/pelvic region of mice. These BAT stores are likely significant metabolic regulators in the mouse and some data suggests that similar BAT depots may also exist in humans.

Regulation of AKT Signaling in Mouse Uterus.

17ß-estradiol (E2) treatment of ovariectomized adult mice stimulates the uterine PI3K-AKT signaling pathway and epithelial proliferation through estrogen receptor 1 (ESR1). However, epithelial proliferation occurs independently of E2/ESR1 signaling in neonatal uteri. Similarly, estrogen-independent uterine epithelial proliferation is seen in adulthood in mice lacking Ezh2, critical for histone methylation, and in wild-type (WT) mice treated neonatally with estrogen. The role of AKT in estrogen-independent uterine epithelial proliferation was the focus of this study. Expression of the catalytically active phosphorylated form of AKT (p-AKT) and epithelial proliferation were high in estrogen receptor 1 knockout and WT mice at postnatal day 6, when E2 concentrations were low, indicating that neither ESR1 nor E2 are essential for p-AKT expression and epithelial proliferation in these mice. However, p-AKT levels and proliferation remained estrogen responsive in preweaning WT mice. Expression of p-AKT and proliferation were both high in uterine luminal epithelium of mice estrogenized neonatally and ovariectomized during adulthood. Increased expression of phosphorylated (inactive) EZH2 was also observed. Consistent with this, Ezh2 conditional knockout mice show ovary-independent uterine epithelial proliferation and high epithelial p-AKT. Thus, adult p-AKT expression is constitutive and E2/ESR1 independent in both model systems. Finally, E2-induced p-AKT expression and normal uterine proliferation did not occur in mice lacking membrane (m)ESR1, indicating a key role for membrane ESR1 in AKT activation. These findings emphasize the importance of AKT activation in promoting uterine epithelial proliferation even when that proliferation is not E2/ESR1 dependent and further indicate that p-AKT can be uncoupled from E2/ESR1 signaling in several experimental scenarios.

Spatial transcriptomics analysis of uterine gene expression in enhancer of zeste homolog 2 conditional knockout mice†.

Histone proteins undergo various modifications that alter chromatin structure, including addition of methyl groups. Enhancer of zeste homolog 2 (EZH2) is a histone methyltransferase that methylates lysine residue 27, and thereby suppresses gene expression. EZH2 plays integral roles in the uterus and other reproductive organs. We have previously shown that conditional deletion of uterine EZH2 results in increased proliferation of luminal and glandular epithelial cells, and RNA-seq analyses reveal several uterine transcriptomic changes in Ezh2 conditional (c) knockout (KO) mice that can affect estrogen signaling pathways. To pinpoint the origin of such gene expression changes, we used the recently developed spatial transcriptomics (ST) method with the hypotheses that Ezh2cKO mice would predominantly demonstrate changes in epithelial cells and/or ablation of this gene would disrupt normal epithelial/stromal gene expression patterns. Uteri were collected from ovariectomized adult WT and Ezh2cKO mice and analyzed by ST. Asb4, Cxcl14, Dio2, and Igfbp5 were increased, Sult1d1, Mt3, and Lcn2 were reduced in Ezh2cKO uterine epithelium vs. WT epithelium. For Ezh2cKO uterine stroma, differentially expressed key hub genes included Cald1, Fbln1, Myh11, Acta2, and Tagln. Conditional loss of uterine Ezh2 also appears to shift the balance of gene expression profiles in epithelial vs. stromal tissue toward uterine epithelial cell and gland development and proliferation, consistent with uterine gland hyperplasia in these mice. Current findings provide further insight into how EZH2 may selectively affect uterine epithelial and stromal compartments. Additionally, these transcriptome data might provide mechanistic understanding and valuable biomarkers for human endometrial disorders with epigenetic underpinnings.

The Roles of the Histone Protein Modifier EZH2 in the Uterus and Placenta.

Epigenetic modifications regulate normal physiological, as well as pathological processes in various organs, including the uterus and placenta. Both organs undergo dramatic and rapid restructuring that depends upon precise orchestration of events. Epigenetic changes that alter transcription and translation of gene-sets regulate such responses. Histone modifications alter the chromatin structure, thereby affecting transcription factor access to gene promoter regions. Binding of histones to DNA is regulated by addition or removal of subunit methyl and other groups, which can inhibit or stimulate transcription. Enhancer of zeste homolog 2 (EZH2) is the catalytic subunit of polycomb repressive complex 2 (PRC2) that catalyzes tri-methylation of histone H3 at Lys 27 (H3K27me3) and subsequently suppresses transcription of genes bound by such histones. Uterine EZH2 expression exerts a critical role in development and function of this organ with deletion of this gene resulting in uterine hyperplasia and expression of cancer-associated transcripts. Elucidating the roles of EZH2 in uterus and placenta is essential as EZH2 dysregulation is associated with several uterine and placental pathologies. Herein, we discuss EZH2 functions in uterus and placenta, emphasizing its physiological and pathological importance.

Mice lacking uterine enhancer of zeste homolog 2 have transcriptomic changes associated with uterine epithelial proliferation.

Enhancer of zeste homolog 2 (EZH2) is a histone methyltransferase that suppresses gene expression. Previously, we developed a conditional null model where EZH2 is knocked out in uterus. Deletion of uterine EZH2 increased proliferation of luminal and glandular epithelial cells. Herein, we used RNA-Seq in wild-type (WT) and EZH2 conditional knockout (Ezh2cKO) uteri to obtain mechanistic insights into the gene expression changes that underpin the pathogenesis observed in these mice. Ovariectomized adult Ezh2cKO mice were treated with vehicle (V) or 17ß-estradiol (E2; 1 ng/g). Uteri were collected at postnatal day (PND) 75 for RNA-Seq or immunostaining for epithelial proliferation. Weighted gene coexpression network analysis was used to link uterine gene expression patterns and epithelial proliferation. In V-treated mice, 88 transcripts were differentially expressed (DEG) in Ezh2cKO mice, and Bmp5, Crabp2, Lgr5, and Sprr2f were upregulated. E2 treatment resulted in 40 DEG with Krt5, Krt15, Olig3, Crabp1, and Serpinb7 upregulated in Ezh2cKO compared with control mice. Transcript analysis relative to proliferation rates revealed two module eigengenes correlated with epithelial proliferation in WT V vs. Ezh2cKO V and WT E2 vs. Ezh2cKO E2 mice, with a positive relationship in the former and inverse in the latter. Notably, the ESR1, Wnt, and Hippo signaling pathways were among those functionally enriched in Ezh2cKO females. Current results reveal unique gene expression patterns in Ezh2cKO uterus and provide insight into how loss of this critical epigenetic regulator assumingly contributes to uterine abnormalities.

The histone methyltransferase EZH2 is required for normal uterine development and function in mice†.

Enhancer of zeste homolog 2 (EZH2) is a rate-limiting catalytic subunit of a histone methyltransferase, polycomb repressive complex, which silences gene activity through the repressive histone mark H3K27me3. EZH2 is critical for epigenetic effects of early estrogen treatment, and may be involved in uterine development and pathologies. We investigated EZH2 expression, regulation, and its role in uterine development/function. Uterine epithelial EZH2 expression was associated with proliferation and was high neonatally then declined by weaning. Pre-weaning uterine EZH2 expression was comparable in wild-type and estrogen receptor 1 knockout mice, showing neonatal EZH2 expression is ESR1 independent. Epithelial EZH2 was upregulated by 17ß-estradiol (E2) and inhibited by progesterone in adult uteri from ovariectomized mice. To investigate the uterine role of EZH2, we developed a EZH2 conditional knockout (Ezh2cKO) mouse using a cre recombinase driven by the progesterone receptor (Pgr) promoter that produced Ezh2cKO mice lacking EZH2 in Pgr-expressing tissues (e.g. uterus, mammary glands). In Ezh2cKO uteri, EZH2 was deleted neonatally. These uteri had reduced H3K27me3, were larger than WT, and showed adult cystic endometrial hyperplasia. Ovary-independent uterine epithelial proliferation and increased numbers of highly proliferative uterine glands were seen in adult Ezh2cKO mice. Female Ezh2cKO mice were initially subfertile, and then became infertile by 9 months. Mammary gland development in Ezh2cKO mice was inhibited. In summary, uterine EZH2 expression is developmentally and hormonally regulated, and its loss causes aberrant uterine epithelial proliferation, uterine hypertrophy, and cystic endometrial hyperplasia, indicating a critical role in uterine development and function.

Mice lacking membrane estrogen receptor 1 are protected from reproductive pathologies resulting from developmental estrogen exposure†.

Both membrane and nuclear fractions of estrogen receptor 1 (ESR1) mediate 17ß-estradiol (E2) actions. Mice expressing nuclear (n)ESR1 but lacking membrane (m)ESR1 (nuclear-only estrogen receptor 1 [NOER] mice) show reduced E2 responsivity and reproductive abnormalities culminating in adult male and female infertility. Using this model, we investigated whether reproductive pathologies caused by the synthetic estrogen diethylstilbestrol (DES) are mitigated by mESR1 ablation. Homozygous and heterozygous wild-type (WT and HET, respectively) and NOER male and female mice were subcutaneously injected with DES (1 mg/kg body weight [BW]) or vehicle daily from postnatal day (PND) 1-5. Uterine histology was assessed in select DES-treated females at PND 5, whereas others were ovariectomized at PND 60 and treated with E2 (10 µg/kg BW) or vehicle 2 weeks later. Neonatal DES exposure resulted in ovary-independent epithelial proliferation in the vagina and uterus of WT but not NOER females. Neonatal DES treatment also induced ovary-independent adult expression of classical E2-induced transcripts (e.g., lactoferrin [Ltf] and enhancer of zeste homolog 2 [Ezh2]) in WT but not NOER mice. At PND 90, DES-treated WT and HET males showed smaller testes and a high incidence of bacterial pyogranulomatous inflammation encompassing the testes, epididymis and occasionally the ductus deferens with spread to lumbar lymph nodes; such changes were largely absent in NOER males. Results indicate that male and female NOER mice are protected from deleterious effects of neonatal DES, and thus mESR1 signaling is required for adult manifestation of DES-induced reproductive pathologies in both sexes.

Membrane-Localized Estrogen Receptor 1 Is Required for Normal Male Reproductive Development and Function in Mice.

Estrogen receptor 1 (ESR1) mediates major reproductive functions of 17ß-estradiol (E2). Male Esr1 knockout (Esr1KO) mice are infertile due to efferent ductule and epididymal abnormalities. The majority of ESR1 is nuclear/cytoplasmic; however, a small fraction is palmitoylated at cysteine 451 in mice and localized to cell membranes, in which it mediates rapid E2 actions. This study used an Esr1 knock-in mouse containing an altered palmitoylation site (C451A) in ESR1 that prevented cell membrane localization, although nuclear ESR1 was expressed. These nuclear-only estrogen receptor 1 (NOER) mice were used to determine the roles of membrane ESR1 in males. Epididymal sperm motility was reduced 85% in 8-month-old NOER mice compared with wild-type controls. The NOER mice had decreased epididymal sperm viability and greater than 95% of sperm had abnormalities, including coiled midpieces and tails, absent heads, and folded tails; this was comparable to 4-month Esr1KO males. At 8 months, daily sperm production in NOER males was reduced 62% compared with controls. The NOER mice had histological changes in the rete testes, efferent ductules, and seminiferous tubules that were comparable with those previously observed in Esr1KO males. Serum T was increased in NOER males, but FSH, LH, and E2 were unchanged. Critically, NOER males were initially subfertile, becoming infertile with advancing age. These findings identify a previously unknown role for membrane ESR1 in the development of normal sperm and providing an adequate environment for spermatogenesis.

Maximal Dexamethasone Inhibition of Luminal Epithelial Proliferation Involves Progesterone Receptor (PR)- and Non-PR-Mediated Mechanisms in Neonatal Mouse Uterus.

Progesterone (P4) and the synthetic glucocorticoid dexamethasone (Dex) inhibit luminal epithelial (LE) proliferation in neonatal mouse uteri. This study determined the roles of progesterone receptor and estrogen receptor 1 (PR and ESR1, respectively) in P4- and Dex-induced inhibition of LE proliferation using PR knockout (PRKO) and Esr1 knockout (Esr1KO) mice. Wild-type (WT), heterozygous, and homozygous PRKO female pups were injected with vehicle, P4 (40 µg/g body weight), or Dex (4 or 40 µg/g body weight) on Postnatal Day 5, then 24 h later immunostained for markers of cell proliferation. In WT and heterozygous mice, P4 sharply reduced LE proliferation, and Dex produced dose-responsive decreases equaling those of P4 at the higher dose. Critically, although both doses of Dex similarly decreased proliferation compared to vehicle-treated PRKOs, treatment of PRKO pups with the high Dex dose (40 µg/g) did not inhibit LE as much as treatments of WT mice with this Dex dose or with P4. Stromal proliferation was stimulated by P4 in WT but not PRKO mice, and Dex did not alter stromal proliferation. Uteri of all genotypes strongly expressed glucocorticoid receptor (GR), demonstrating that impaired Dex effects in PRKOs did not reflect GR deficiency. Furthermore, inhibition of LE proliferation by Dex (40 µg/g body weight) in Esr1KO mice was normal, so this process does not involve ESR1. In summary, inhibitory Dex effects on LE proliferation occur partially through non-PR-mediated mechanisms, presumably GR, as indicated by Dex inhibition of LE proliferation in PRKOs. However, maximal inhibitory Dex effects on uterine LE proliferation are not seen in PRKO mice with even high Dex, indicating that maximal Dex effects in WT mice also involve PR.

Neonatal uterine and vaginal cell proliferation and adenogenesis are independent of estrogen receptor 1 (ESR1) in the mouse.

Neonatal uterus and vagina express estrogen receptor 1 (ESR1) and respond mitogenically to exogenous estrogens. However, neonatal ovariectomy does not inhibit preweaning uterine cell proliferation, indicating that this process is estrogen independent. Extensive literature suggests that ESR1 can be activated by growth factors in a ligand-independent manner and drive uterine cell proliferation. Alternatively, neonatal uterine cell proliferation could be ESR1 independent despite its obligatory role in adult luminal epithelial proliferation. To determine ESR1's role in uterine and vaginal development, we analyzed cell proliferation, apoptosis, and uterine gland development (adenogenesis) in wild-type (WT) and Esr1 knockout (Esr1KO) mice from Postnatal Day 2 to Postnatal Day 60. Uterine and vaginal cell proliferation, apoptosis, and uterine adenogenesis were comparable in WT and Esr1KO mice before weaning. By Days 29-60, glands had regressed, and uterine cell proliferation was reduced in Esr1KO mice in contrast to continued adenogenesis and proliferation in WT. Apoptosis in Esr1KO uterine epithelium was not increased compared to WT at any age, indicating that differences in cell proliferation, rather than apoptosis, cause divergence of uterine size in these two groups at puberty. Similarly, vaginal epithelial proliferation was reduced, and the epithelium became atrophic in Esr1KO mice by 29 days of age and later in Esr1KO mice. These results indicate that preweaning uterine and vaginal development is ESR1 independent but becomes dependent on ESR1 by Day 29 on. It is not yet clear what mechanisms drive preweaning vaginal and uterine development, but ligand-independent activation of ESR1 is not involved.

Plasticity of spermatogonial stem cells.

There have been significant breakthroughs over the past decade in the development and use of pluripotent stem cells as a potential source of cells for applications in regenerative medicine. It is likely that this methodology will begin to play an important role in human clinical medicine in the years to come. This review describes the plasticity of one type of pluripotent cell, spermatogonial stem cells (SSCs), and their potential therapeutic applications in regenerative medicine and male infertility. Normally, SSCs give rise to sperm when in the testis. However, both human and murine SSCs can give rise to cells with embryonic stem (ES) cell-like characteristics that can be directed to differentiate into tissues of all three embryonic germ layers when placed in an appropriate inductive microenvironment, which is in contrast to other postnatal stem cells. Previous studies have reported that SSCs expressed an intermediate pluripotent phenotype before differentiating into a specific cell type and that extended culture was necessary for this to occur. However, recent studies from our group using a tissue recombination model demonstrated that SSCs differentiated rapidly into another tissue, in this case, prostatic epithelium, without expression of pluripotent ES cell markers before differentiation. These results suggest that SSCs are capable of directly differentiating into other cell types without going through an intermediate ES cell-like stage. Because SSCs do not require reprogramming to achieve a pluripotent state, they are an attractive source of pluripotent cells for use in regenerative medicine.

Antibody-specific detection of CAIX in breast and prostate cancers.

Carbonic anhydrase IX (CAIX) is frequently expressed in human tumors and serves as a marker for hypoxia. Further, CAIX expression is considered a predictor of poor survival in many, but not all, cancer types. Herein, we compare the specificity of two CAIX antibodies: the M75, monoclonal antibody which recognizes an epitope in the N-terminus and a commercially available polyclonal antibody generated against a C-terminal peptide (NB100-417). Western blot analysis of multiple breast cell lines revealed that the polyclonal antibody detected both membrane-bound and soluble proteins. The M75 antibody recognized only the membrane-bound species, which is presumed to be CAIX. These data were confirmed in an aggressive prostate cell line. We further compared these antibodies in prostate tumors by immunohistochemistry. Staining with NB100 was comparable to that of the M75 antibody, but only at high dilution. Otherwise, cytoplasmic staining was also noted. Two-dimensional gel electrophoresis followed by mass spectrometric analysis revealed that the cytoplasmic protein detected by NB100 is beta-tubulin. This cross-reactivity could lead to false-positives for CAIX expression in samples where cytosolic proteins are present.

Downregulation of BCL-2 induces downregulation of carbonic anhydrase IX, vascular endothelial growth factor, and pAkt and induces radiation sensitization.

OBJECTIVES: Our group previously demonstrated that expression of the oncogene, Bcl-2, was associated with radiation resistance. The aim of the present study was to determine whether Bcl-2 expression in radiation-resistant tumors was associated with expression of carbonic anhydrase IX (CAIX), vascular endothelial growth factor (VEGF), and pAkt and whether downregulation of Bcl-2 could modulate the expression of CAIX, VEGF, and pAkt. METHODS: Two prostate cancer cell lines, PC-3-Bcl-2 and PC-3-Neo, were injected into the subcutaneous flanks of 192 athymic male nude mice; 96 received PC-3 Bcl-2 and 96 PC-3-Neo. The mice were then treated with antisense Bcl-2 oligodeoxynucleotide (ASODN), reverse control ODN, or vehicle only (mock treatment) with or without irradiation (4 Gy). The tumors were monitored for growth (ie, volume) over time, resected at various points, and processed and sectioned for protein analyses. RESULTS: Bcl-2 ASODN treatment was associated with downregulation of Bcl-2, VEGF, pAkt, and CAIX. PC-3-Bcl-2 and PC-3-Neo prostate tumor xenografts in mice treated with the combination of Bcl-2 ASODN and irradiation were significantly (ie, one third) smaller than those in mice treated with reverse control ODN alone, Bcl-2 ASODN alone, irradiation alone, or reverse control ODN plus irradiation (P = 0.0001). An increased tumor response to the combined therapy was associated with decreased expression of Bcl-2, VEGF, and pAkt proteins. CONCLUSIONS: These findings have demonstrated an intimate relationship among CAIX, VEGF, pAkt, and Bcl-2 in prostate tumors. Thus, CAIX might prove effective as a potential marker of tumor radiation resistance. Downregulation of CAIX might lead to radiation sensitization. Consequently, the combination of CAIX reduction and radiotherapy warrants further consideration as a new strategy for therapy.

Soy isoflavones exert differential effects on androgen responsive genes in LNCaP human prostate cancer cells.

The high consumption of soy isoflavones in Asian diets has been correlated to a lower incidence of clinically important cases of prostate cancer. This study characterized the effects of a soy-derived isoflavone concentrate (ISF) on growth and gene expression profiles in the LNCaP, an androgen-sensitive human prostate cancer cell line. ISF caused a dose-dependent decrease in viability (P < 0.05) and DNA synthesis (P < 0.01), as well as an accumulation of cells in G(2)/M, and G(0)/G(1) phases of the cell cycle compared with controls. Using Affymetrix oligonucleotide DNA microarrays (U133A), we determined that ISF upregulated 80 genes and downregulated 33 genes (P < 0.05) involving androgen-regulated genes and pathways controlling cell cycle, metabolism, and intracellular trafficking. Changes in the expression of the genes of interest, identified by microarrays, were validated by Western immunoblot, Northern blot, and luciferase reporter assays. Prostate-specific antigen, homeobox protein NKX3, and cyclin B mRNA were significantly reduced, whereas mRNA was significantly upregulated for p21(CIP1), a major cell cycle inhibitory protein, and fatty acid and cholesterol synthesis pathway genes. ISF also significantly increased cyclin-dependent kinase inhibitor p27(KIP1) and FOXO3A/FKHRL1, a forkhead transcription factor. A differential pattern of androgen-regulated genes was apparent with genes involved in prostate cancer progression being downregulated by ISF, whereas metabolism genes were upregulated. In summary, we found that ISF inhibits the growth of LNCaP cells through the modulation of cell cycle progression and the differential expression of androgen-regulated genes. Thus, ISF treatment serves to identify new therapeutic targets designed to prevent proliferation of malignant prostate cells.

Soy isoflavones alter expression of genes associated with cancer progression, including interleukin-8, in androgen-independent PC-3 human prostate cancer cells.

High consumption of soy isoflavones in Asian diets has been correlated with a lower incidence of clinically important cases of prostate cancer. The chemopreventive properties of these diets may result from an interaction of several types of isoflavones, including genistein and daidzein. The present study investigated the effects of a soy isoflavone concentrate (ISF) on growth and gene expression profiles of PC-3 human prostate cancer cells. Trypan blue exclusion and [3H]-thymidine incorporation assays showed that ISF decreased cell viability and caused a dose-dependent inhibition of DNA synthesis, respectively, with 50% inhibition (IC50) of DNA synthesis at 52 mg/L (P = 0.05). The glucoside conjugates of genistein and daidzein in ISF were converted to bioactive free aglycones in cell culture in association with the inhibition of DNA synthesis. Flow cytometry and Western immunoblot analyses showed that ISF at 200 mg/L caused an accumulation of cells in the G2/M phase of the cell cycle (P < 0.05) and decreased cyclin A by 20% (P < 0.05), respectively. The effect of ISF on the gene expression profile of PC-3 cells was analyzed using Affymetrix oligonucleotide DNA microarrays that interrogate approximately 17,000 human genes. Of the 75 genes altered by ISF, 28 were upregulated and 47 were downregulated (P < 0.05). Further analysis showed that IL-8, matrix metalloproteinase 13, inhibin beta A, follistatin, and fibronectin mRNA levels were significantly reduced, whereas the expression of p21(CIP1), a major cell cycle inhibitory protein, was increased. The effects of ISF on the expression of IL-8 and p21(CIP1) mRNA and protein were validated at high and low ISF concentrations. Our data show that ISF inhibits the growth of PC-3 cells through modulation of cell cycle progression and the expression of genes involved in cell cycle regulation, metastasis, and angiogenesis.

Mechanisms of the growth inhibitory effects of the isoflavonoid biochanin A on LNCaP cells and xenografts.

BACKGROUND: Isoflavones inhibit the growth of some types of tumor cells, including prostate adenocarcinoma. This study used LNCaP cells and xenografts to investigate the mechanisms of the antiproliferative effects of biochanin A, a major isoflavone present in red clover but not soy-derived products. METHODS: LNCaP cells were exposed to varying doses of biochanin A to evaluate viability, DNA synthesis, and DNA fragmentation (TUNEL) analysis. Regulation of gene expression was determined by using Western immunoblotting and cDNA microarrays. Anti-tumorigenic effects were evaluated by using athymic mice with LNCaP flank tumors. RESULTS: Biochanin A induced a dose-dependent inhibition of proliferation and [(3)H]thymidine incorporation that correlated with increased DNA fragmentation, indicative of apoptosis. Western blot analyses of cell cycle regulatory proteins revealed that biochanin A significantly decreased expression of cyclin B and p21, whereas flow cytometry showed that cells were accumulating in the G(0)/G(1) phase. cDNA microarray analyses identified 29 down-regulated genes with six reduced below assay detection limits. Eleven genes were up-regulated, including 9 that were undetectable in controls. In mice with LNCaP xenografts, biochanin A significantly reduced tumor size and incidence. CONCLUSION: These results indicate that biochanin A inhibits prostate cancer cell growth through induction of cell cycle arrest and apoptosis. Biochanin A-regulated genes suggest multiple pathways of action. Biochanin A inhibits the incidence and growth of LNCaP xenograft tumors in athymic mice.

Benzo(a)pyrene, but not 2,3,7,8-tetrachlorodibenzo-p-dioxin, alters cell adhesion proteins in human uterine RL95-2 cells.

This study compared the effects of benzo(a)pyrene (BaP) and 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), two aryl hydrocarbon receptor agonists, on cell attachment and adherens junction proteins in RL95-2 human uterine endometrial cells. Exposure to 10 microM BaP significantly decreased cell attachment to Matrigel, whereas 10 nM TCDD had no effect. Immunocytochemistry and Western immunoblot analysis showed that BaP, but not TCDD, produced a marked loss of plasma membrane epidermal growth factor receptor (EGF-R) localized along intercellular boundaries. BaP-treated cells exhibited significant decreases in beta-catenin and cadherin protein levels, while vinculin levels remained unchanged relative to control. In contrast, TCDD treatment had no effect on the levels of beta-catenin, cadherin, or vinculin. Further studies using the fluorescein labeled peptide phalloidin showed the presence of continuous subcortical actin filaments in control cells, whereas BaP-treated cells had subcortical actin aggregates. Thus, in contrast to TCDD, BaP produces a loss of cell attachment involving decreased localization of molecules important for cell-cell interactions in RL95-2 cells.