ABSTRACT
BACKGROUND: Prostate cancer is an important public health problem. It is an excellent candidate disease for chemoprevention because prostate cancer is typically slow growing and is usually diagnosed in elderly males. Pygeum africanum (Prunus africana or Rosaceae) is an African prune (plum) tree found in tropical Africa. An extract from the bark of Pygeum africanum has been used in Europe as a prevention and treatment of prostate disorders including benign prostatic hypertrophy (BPH). More recently in the USA, the phytotherapeutic preparations of Pygeum africanum and Saw palmetto have been marketed for prostate health including prostate cancer prevention and treatment. METHODS: The anti-cancer potential of Pygeum africanum has been tested both in vitro (PC-3 and LNCaP cells) and in vivo (TRAMP mouse model). RESULTS: In tissue culture, ethanolic extracts (30%) of Pygeum africanum inhibited the growth of PC-3 and LNCaP cells; induced apoptosis and altered cell kinetics; down regulated ERalpha and PKC-alpha protein, and demonstrated good binding ability to both mouse uterine estrogen receptors and LNCaP human androgen receptors. TRAMP mice fed Pygeum africanum showed a significant reduction (P = 0.034) in prostate cancer incidence (35%) compared to casein fed mice (62.5%). CONCLUSION: Pygeum africanum, which is widely used in Europe and USA for treatment of BPH, has a significant role in regulation of prostate cancer both in vitro and in vivo and therefore may be a useful supplement for people at high risk for developing prostate cancer.
Subject(s)
Antineoplastic Agents/therapeutic use , Carcinoma/drug therapy , Phytosterols/therapeutic use , Phytotherapy , Prostatic Neoplasms/drug therapy , Prunus africana , Animals , Cell Line, Tumor , Drug Evaluation, Preclinical , Humans , Male , Mice , Plant Extracts/therapeutic useABSTRACT
Estrogen facilitates baroreflex heart rate responses evoked by intravenous infusion of ANG II and phenylephrine (PE) in ovariectomized female mice. The present study aims to identify the estrogen receptor subtype involved in mediating these effects of estrogen. Baroreflex responses to PE, ANG II, and sodium nitroprusside (SNP) were tested in intact and ovariectomized estrogen receptor-alpha knockout (ERalphaKO) with (OvxE+) or without (OvxE-) estrogen replacement. Wild-type (WT) females homozygous for the ERalpha(+/+) were used as controls. Basal mean arterial pressures (MAP) and heart rates were comparable in all the groups except the ERalphaKO-OvxE+ mice. This group had significantly smaller resting MAP, suggesting an effect of estrogen on resting vascular tone possibly mediated by the ERbeta subtype. Unlike the WT females, estrogen did not facilitate baroreflex heart rate responses to either PE or ANG II in the ERalphaKO-OvxE+ mice. The slope of the line relating baroreflex heart rate decreases with increases in MAP evoked by PE was comparable in ERalphaKO-OvxE- (-6.97 +/- 1.4 beats.min(-1).mmHg(-1)) and ERalphaKO-OvxE+ (-6.18 +/- 1.3) mice. Likewise, the slope of the baroreflex bradycardic responses to ANG II was similar in ERalphaKO-OvxE- (-3.87 +/- 0.5) and ERalphaKO-OvxE+(-2.60 +/- 0.5) females. Data suggest that estrogen facilitation of baroreflex responses to PE and ANG II is predominantly mediated by ERalpha subtype. A second important observation in the present study is that the slope of ANG II-induced baroreflex bradycardia is significantly blunted compared with PE in the intact as well as the ERalphaKO-OvxE+ females. We have previously reported that this ANG II-mediated blunting of cardiac baroreflexes is observed only in WT males and not in ovariectomized WT females independent of their estrogen replacement status. The present data suggest that in females lacking ERalpha, ANG II causes blunting of cardiac baroreflexes similar to males and may be indicative of a direct modulatory effect of the ERalpha on those central mechanisms involved in ANG II-induced resetting of cardiac baroreflexes. These observations suggest an important role for ERalpha subtype in the central modulation of baroreflex responses. Lastly, estrogen did not significantly affect reflex tachycardic responses to SNP in both WT and ERalphaKO mice.
Subject(s)
Baroreflex/drug effects , Estrogen Receptor alpha/genetics , Estrogen Receptor alpha/metabolism , Estrogens/pharmacology , Heart Rate/drug effects , Angiotensin II/pharmacology , Animals , Baroreflex/physiology , Blood Pressure/drug effects , Blood Pressure/physiology , Consciousness , Female , Heart Rate/physiology , Mice , Mice, Inbred C57BL , Mice, Knockout , Nitroprusside/pharmacology , Ovariectomy , Ovary/physiology , Phenylephrine/pharmacology , Vasoconstrictor Agents/pharmacology , Vasodilator Agents/pharmacologyABSTRACT
Primary evidence for novel estrogen signaling pathways is based upon well-documented estrogenic responses not inhibited by estrogen receptor antagonists. In addition to 17beta-E2, the catechol estrogen 4-hydroxyestradiol (4OHE2) has been shown to elicit biological responses independent of classical estrogen receptors in estrogen receptor-alpha knockout (ERalphaKO) mice. Consequently, our research was designed to biochemically characterize the protein(s) that could be mediating the biological effects of catechol estrogens using enzymatically synthesized, radiolabeled 4-hydroxyestrone (4OHE1) and 4OHE2. Scatchard analyses identified a single class of high-affinity (K(d) approximately 1.6 nM), saturable cytosolic binding sites in several ERalphaKO estrogen-responsive tissues. Specific catechol estrogen binding was competitively inhibited by unlabeled catechol estrogens, but not by 17beta-E2 or the estrogen receptor antagonist ICI 182,780. Tissue distribution studies indicated significant binding differences both within and among various tissues in wild-type, ERalphaKO, and aromatase knockout female mice. Ligand metabolism experiments revealed extensive metabolism of labeled catechol estrogen, suggesting that catechol estrogen metabolites were responsible for the specific binding. Collectively, our data provide compelling evidence for the interaction of catechol estrogen metabolites with a novel binding protein that exhibits high affinity, specificity, and selective tissue distribution. The extensive biochemical characterization of this binding protein indicates that this protein may be a receptor, and thus may mediate ERalpha/beta-independent effects of catechol estrogens and their metabolites.
Subject(s)
Aromatase/genetics , Estradiol/analogs & derivatives , Estrogens, Catechol/metabolism , Proteins/metabolism , Receptors, Estrogen/genetics , Animals , Aromatase/metabolism , Binding, Competitive , Estradiol/chemical synthesis , Estradiol/isolation & purification , Estradiol/metabolism , Estrogen Antagonists/metabolism , Estrogen Receptor alpha , Female , Fulvestrant , Hydroxyestrones/chemical synthesis , Hydroxyestrones/isolation & purification , Hydroxyestrones/metabolism , Kidney/metabolism , Liver/metabolism , Male , Mice , Mice, Knockout , Ovary/metabolism , Receptors, Estrogen/metabolism , Substrate Specificity , Tissue Distribution , Uterus/metabolismABSTRACT
Leydig cells, which produce the primary male steroid hormone testosterone (T), express the two estrogen receptor (ER) subtypes, ERalpha and ERbeta, and have the capacity to convert testosterone to the natural estrogen 17beta-estradiol. Thus, Leydig cells are subject to estrogen action. The development of transgenic mice that are homozygous for targeted deletion of genes encoding the ER subtypes provides an opportunity to examine the role of estrogen in Leydig cell function. In this study androgen biosynthesis was analyzed in Leydig cells from mice that were homozygous for targeted deletion of the ERalpha gene (alphaERKO). T production by alphaERKO Leydig cells was 2-fold higher than that in wild-type (WT) cells. Serum T levels were accordingly higher in alphaERKO compared with WT mice (5.1 +/- 1.1 vs. 2.2 +/- 0.4 ng/ml; P = 0.01) as were serum LH levels (1.31 +/- 0.3 vs. 0.45 +/- 0.08 ng/ml; P = 0.01). Mice that were treated with the pure antiestrogen ICI 182,780 at 100 micro g/kg.d for 7 d, effectively abrogating ER-mediated activity, also had 2-fold elevations in the serum levels of LH (1.15 +/- 0.3 vs. 0.45 +/- 0.2 ng/ml) and T (4.3 +/- 1.1 vs. 2.2 +/- 0.2 ng/ml; P = 0.01). Increased androgen biosynthesis by alphaERKO Leydig cells was associated with higher steroidogenic enzyme activity, especially of cytochrome P450 17alpha-hydroxylase/17-20 lyase (P450(17alpha)) and 17beta-hydroxysteroid dehydrogenase (17beta-HSD), as measured by conversion of radiolabeled steroid substrates to T or its precursors. The largest increases in enzymatic activity were observed for P450(17alpha) (423 +/- 45 pmol/min.10(6) cells in alphaERKO Leydig cells vs. 295 +/- 27 pmol/min.10(6) cells in WT cells; P < 0.01). Consistent with steroidogenic enzyme activity, the testis of alphaERKO mice expressed higher steady state mRNA levels for steroidogenic acute regulatory protein and two enzymes involved in androgen biosynthesis, P450(17alpha) and 17beta-HSD type III, as determined by semiquantitative RT-PCR. Compared with the controls, higher steady state mRNA levels for steroidogenic acute regulatory protein and P450(17alpha) were also measured in the testis of ICI 182,780-treated mice. In a second set of experiments estrogen administration reduced serum LH and T levels in WT controls, whereas alphaERKO mice were unaffected. Although exposure of WT and alphaERKO Leydig cells to estrogen in vitro did not affect androgen biosynthesis, incubation with ICI 182,780 reduced T production by WT, but not alphaERKO, Leydig cells. These observations indicate that abrogation of the ERalpha gene by targeted deletion or treatment with an antiestrogen increases Leydig cell steroidogenesis in association with elevations in the serum levels of LH, which presumably is the result of estrogen insensitivity at the level of the hypothalamus and/or pituitary gonadotropes. Furthermore, the decrease in T production by WT Leydig cells and not alphaERKO Leydig cells occasioned by incubation with ICI 182,780 suggests that of the ER subtypes, ERalpha has a regulatory role in Leydig cell steroidogenic function.
Subject(s)
Androgens/biosynthesis , Estradiol/analogs & derivatives , Leydig Cells/metabolism , Receptors, Estrogen/deficiency , 17-Hydroxysteroid Dehydrogenases/metabolism , Animals , Estradiol/pharmacology , Estrogen Antagonists/pharmacology , Estrogen Receptor alpha , Fulvestrant , Luteinizing Hormone/blood , Male , Mice , Mice, Knockout , Receptors, Estrogen/genetics , Receptors, Estrogen/physiology , Reverse Transcriptase Polymerase Chain Reaction , Steroid 17-alpha-Hydroxylase/metabolism , Testosterone/biosynthesis , Testosterone/bloodABSTRACT
In this study we examine the effect of the phytoestrogen genistein on DNA methylation. DNA methylation is thought to inhibit transcription of genes by regulating alterations in chromatin structure. Estrogenic compounds have been reported to regulate DNA methylation in a small number of studies. Additionally, phytoestrogens are believed to affect progression of some human diseases, such as estrogen-dependent cancers, osteoporosis and cardiovascular disease. Specifically, our working hypothesis is that certain soy phytoestrogens, such as genistein, may be involved in preventing the development of certain prostate and mammary cancers by maintaining a protective DNA methylation profile. The objective of the present study is to use mouse differential methylation hybridization (DMH) arrays to test for changes in the methylation status of the cytosine guanine dinucleotide (CpG) islands in the mouse genome by examining how these methylation patterns are affected by genistein. Male mice were fed a casein-based diet (control) or the same diet containing 300 mg genistein/kg according to one of four regimens: control diet for 4 wk, genistein diet for 4 wk, control diet for 2 wk followed by genistein diet for 2 wk and genistein diet for 2 wk followed by control diet for 2 wk. DNA from liver, brain and prostate were then screened with DMH arrays. Clones with methylation differences were sequenced and compared with known sequences. In conclusion, consumption of genistein diet was positively correlated with changes in prostate DNA methylation at CpG islands of specific mouse genes.