Horacio Cardenas

Epigenetic targeting of ovarian cancer stem cells

Emerging results indicate that cancer stem-like cells contribute to chemoresistance and poor clinical outcomes in many cancers, including ovarian cancer. As epigenetic regulators play a major role in the control of normal stem cell differentiation, epigenetics may offer a useful arena to develop strategies to target cancer stem-like cells. Epigenetic aberrations, especially DNA methylation, silence tumor-suppressor and differentiation-associated genes that regulate the survival of ovarian cancer stem-like cells (OCSC). In this study, we tested the hypothesis that DNA-hypomethylating agents may be able to reset OCSC toward a differentiated phenotype by evaluating the effects of the new DNA methytransferase inhibitor SGI-110 on OCSC phenotype, as defined by expression of the cancer stem-like marker aldehyde dehydrogenase (ALDH). We demonstrated that ALDH(+) ovarian cancer cells possess multiple stem cell characteristics, were highly chemoresistant, and were enriched in xenografts residual after platinum therapy. Low-dose SGI-110 reduced the stem-like properties of ALDH(+) cells, including their tumor-initiating capacity, resensitized these OCSCs to platinum, and induced reexpression of differentiation-associated genes. Maintenance treatment with SGI-110 after carboplatin inhibited OCSC growth, causing global tumor hypomethylation and decreased tumor progression. Our work offers preclinical evidence that epigenome-targeting strategies have the potential to delay tumor progression by reprogramming residual cancer stem-like cells. Furthermore, the results suggest that SGI-110 might be administered in combination with platinum to prevent the development of recurrent and chemoresistant ovarian cancer.

Flavone deglycosylation increases their anti-inflammatory activity and absorption

SCOPE Flavones have reported anti-inflammatory activities, but the ability of flavone-rich foods to reduce inflammation is unclear. Here, we report the effect of flavone glycosylation in the regulation of inflammatory mediators in vitro and the absorption of dietary flavones in vivo. METHODS AND RESULTS The anti-inflammatory activities of celery extracts, some rich in flavone aglycones and others rich in flavone glycosides, were tested on the inflammatory mediators tumor necrosis factor α (TNF-α) and nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) in lipopolysaccharide-stimulated macrophages. Pure flavone aglycones and aglycone-rich extracts effectively reduced TNF-α production and inhibited the transcriptional activity of NF-κB, while glycoside-rich extracts showed no significant effects. Deglycosylation of flavones increased cellular uptake and cytoplasmic localization as shown by high-performance liquid chromatography (HPLC) and microscopy using the flavonoid fluorescent dye diphenylboric acid 2-aminoethyl ester (DPBA). Celery diets with different glycoside or aglycone contents were formulated and absorption was evaluated in mice fed with 5 or 10% celery diets. Relative absorption in vivo was significantly higher in mice fed with aglycone-rich diets as determined by HPLC-MS/MS (where MS/MS is tandem mass spectrometry). CONCLUSION These results demonstrate that deglycosylation increases absorption of dietary flavones in vivo and modulates inflammation by reducing TNF-α and NF-κB, suggesting the potential use of functional foods rich in flavones for the treatment and prevention of inflammatory diseases.

Attenuation of Estrogenic Effects by Dihydrotestosterone in the Pig Uterus Is Associated with Downregulation of the Estrogen Receptors

Abstract Androgens are known to attenuate some effects of estradiol-17β (E) in the uterus. The objectives of the present experiment were to determine effects of 5α-dihydrotestosterone (DHT) on estrogenic actions in the pig uterus and its associations with changes in expression of the estrogen receptor (ER) α and ERβ. Postpubertal gilts (120–130 kg of body weight; n = 16) were ovariectomized, and 3–4 weeks later received once-a-day injections (i.m.) of one of the following treatments during four consecutive days: 1) vehicle (corn oil), 2) E (250 μg), 3) E (250 μg) plus 1 mg DHT, or 4) E (250 μg) plus 10 mg DHT. Uterine tissues were collected 24 h after the last treatment. Gilts receiving E or E plus 1 mg DHT had greater uterine wet weight, uterine horn diameter, luminal epithelium thickness, and endometrial gland diameter compared with gilts treated with vehicle or E plus 10 mg DHT. Gilts receiving E or E plus 1 mg DHT were not different in these characteristics. Relative amounts of mRNAs in the endometrium for the cell proliferation marker histone H2a and the E-inducible protein complement component C3 increased in gilts treated with E compared with gilts treated with vehicle. E-induced increases in histone H2a and C3 mRNAs were not altered by cotreatment with E plus 1 mg DHT but were inhibited by E plus 10 mg DHT. Androgen receptor (AR) mRNA in the endometrium increased by treatment with E. Cotreatment of gilts with E and DHT did not alter the E-induced AR mRNA increase. Gilts treated with E plus 10 mg DHT had lesser amounts of immunoreactive ERα in cell nuclei of the myometrium and endometrial stroma and a tendency for a decrease in luminal epithelium compared with gilts treated with E. Amounts of immunoreactive ERα in glandular epithelium were not influenced by the treatments. Relative amounts of ERα and ERβ mRNAs decreased in the endometrium of gilts treated with E plus 10 mg DHT compared with gilts treated with E. Downregulation of the ERs, particularly ERα in the myometrium and endometrial stroma, might be a relevant mechanism in the antagonism of estrogenic effects by DHT in the pig uterus.

Estrogen Receptor β in the Sheep Ovary During the Estrous Cycle and Early Pregnancy

Abstract Objectives were to sequence and examine the expression of the estrogen receptor β (ERβ) in the sheep ovary. The sequence of the ovine ERβ (oERβ) was determined using reverse-transcription polymerase chain reaction (RT-PCR) and cloning techniques. The reading frame of oERβ contained 527 amino acids and exhibited high overall homology with cow (98%), rat (88%), and human (88%) ERβ. In addition, an oERβ isoform having a 139-base pair deletion (oERβ1) was identified. The predicted amino acid sequence of this isoform is lacking the ligand-binding and carboxyl-terminal transactivation domains. The oERβ protein and mRNA were determined in ovaries obtained from ewes on Days 0 (first day of estrus), 2, 6, and 10 of the estrous cycle and Day 30 of gestation. Immunohistochemistry showed that oERβ protein was located in granulosa cells, the ovarian surface epithelium, endothelium, and Day 2 corpus luteum (CL). Weak immunostaining for ERβ was detected in the theca interna. Relative steady-state amounts of oERβ mRNA in the CL were determined using semiquantitative RT-PCR. Amounts of oERβ mRNA were greater (P < 0.05) during CL formation (Day 2) than at later stages. The oERβ to oERβ1 mRNA ratio was lower (P < 0.05) on Day 2 than on Day 10 or Day 30 due to a decrease in amounts of oERβ1. Results indicate that the oERβ is a 527-amino acid protein expressed in specific cells of the ovary. Changes in relative amounts of full-length oERB and a deletion isoform in CL occurred during the estrous cycle, suggesting that these two types of ERβ might regulate estrogen actions during early CL development in sheep.

Cortisol and prolactin concentrations during three different seasons in relocated Brahman and Hereford bulls

The objective of this study was to evaluate seasonal changes of cortisol and prolactin (PRL) concentrations in Brahman and Hereford bulls moved to locations that differ in geographical and environmental conditions. Postpubertal Hereford bulls from Montana (n = 15) and Nebraska (n = 15) and Brahman bulls from Texas (n = 18) were located in or relocated to Montana, Nebraska or Texas so that each location had 5 Montana Herefords, 5 Nebraska Herefords and 6 Texas Brahman bulls. Blood samples were collected at 20-minute intervals for 8 hours in November (Fall 1), April (Spring) and November (Fall 2) of the next year. These dates corresponded to 6, 12 and 18 months, respectively, after relocation in May of the first year. Cortisol concentrations were higher (P<0.05) in Fall 1 than in Fall 2 and were higher (P<0.05) for bulls in Montana than for bulls in Texas. The decrease in cortisol concentrations from Fall 1 to Fall 2 was negatively related (P<0.05) to age and weight. There was a three-way interaction (P<0.05) of breed-type origin, location and season for PRL concentrations. Seasonal patterns of PRL concentrations differed between relocated Texas Brahman and Hereford bulls, and patterns for relocated bulls differed from those of the nonrelocated bulls. Seasonal patterns of PRL were influenced to a greater extent by relocation in Texas Brahman bulls than in Hereford bulls.

Dose-response relationships of exogenous progesterone shortly after ovulation on estrous cycle length, blastocyst development and fertility in sheep

Maternal recognition of pregnancy is a period of competing signals; a luteolytic signal from the endometrium and an antiluteolytic signal from the blastocyst (s). Exogenous progesterone indirectly enhances these signals, causing premature release of prostaglandin and stimulating growth of blastocysts. In Experiment 1, 78 ewes were injected with vehicle or varying dosages of progesterone on Days 2–4 (Day 0 represents onset of estrus) for the purpose of comparing the minimal dose that shortened the estrous cycle, in non-pregnant ewes, to the minimal dose that enhanced growth of blastocysts to Day 13, in pregnant ewes. In Experiment 2, a field trial was conducted using Targhee and Polypay ewes to evaluate their fertility after treatment with vehicle or 6 mg of progesterone (n = 55). Analysis of plasma samples indicated that none of the dosages of exogenous progesterone altered concentrations of progesterone by Days 5 or 10, suggesting that treatment with exogenous progesterone failed to alter luteal function to Day 10. The minimal dose of progesterone that shortened (P<0.05) the estrous cycle was 6 mg and was the same dose that began to stimulate (P<0.05) blastocyst growth. Lambing rates of Targhee ewes were not different following treatment with exogenous progesterone. However, the lambing rate of Polypay ewes increased (P<0.05) from 200 to 256%. These data suggest that treatment of sheep, predisposed to an ovulation rate greater than two, with progesterone improved embryonic survival by a still unknown mechanism(s) that might have also advanced luteolytic and antiluteolytic signals.

EZH2 inhibition promotes epithelial-to-mesenchymal transition in ovarian cancer cells

Cancer cells acquire essential characteristics for metastatic dissemination through the process of epithelial-to-mesenchymal transition (EMT), which is regulated by gene expression and chromatin remodeling changes. The enhancer of zeste homolog 2 (EZH2), the catalytic subunit of the polycomb repressive complex 2 (PRC2), catalyzes trimethylation of lysine 27 of histone H3 (H3K27me3) to repress gene transcription. Here we report the functional roles of EZH2-catalyzed H3K27me3 during EMT in ovarian cancer (OC) cells. TGF-β-induced EMT in SKOV3 OC cells was associated with decreased levels of EZH2 and H3K27me3 (P<0.05). These effects were delayed (~72 h relative to EMT initiation) and coincided with increased (>15-fold) expression of EMT-associated transcription factors ZEB2 and SNAI2. EZH2 knockdown (using siRNA) or enzymatic inhibition (by GSK126) induced EMT-like changes in OC cells. The EMT regulator ZEB2 was upregulated in cells treated with either approach. Furthermore, TGF-β enhanced expression of ZEB2 in EZH2 siRNA- or GSK126-treated cells (P<0.01), suggesting that H3K27me3 plays a role in TGF-β-stimulated ZEB2 induction. Chromatin immunoprecipitation assays confirmed that TGF-β treatment decreased binding of EZH2 and H3K27me3 to the ZEB2 promoter (P<0.05). In all, these results demonstrate that EZH2, by repressing ZEB2, is required for the maintenance of an epithelial phenotype in OC cells.

Effect of the rate of progesterone decline at luteolysis on the ovulatory follicles and subsequent estrous cycle length in ewes

Follicular and interestrous characteristics were examined in 34 ewes after experiencing either a rapid decline in plasma concentrations of progesterone at luteolysis [prostaglandin F2 alpha (PGF2 alpha)-induced] or a slow rate of decline, lasting over 72 h. All ewes were given PGF2 alpha on day 10 (day 0 = estrus). A slow rate of decline was established in 17 ewes by the intravenous infusion of progesterone initially at 72 ml h-1, delivering 4.5 mg progesterone h-1, then decreasing the infusion rate by 1 ml h-1 for the next three days. Seventeen additional ewes, predestined to experience a rapid decline in progesterone, were infused with vehicle. In Experiment 1, after infusion, ewes (6 ewes/group) were necropsied at the onset of estrus and follicle diameter was determined, follicular fluid was aspirated and the remaining follicular wall was microscopically examined to determine the number of granulosa cell layers. In Experiment 2, the interestrous interval, after infusion, was observed in both groups of ewes (11 ewes/group). Ewes experiencing a rapid rate of progesterone decline at luteolysis had no differences in follicle diameter nor follicular concentration of progesterone or estradiol but their ovulatory follicles contained fewer (P < 0.01) granulosa cell layers and the resulting estrous cycle was longer (P < 0.05) than ewes experiencing a slow rate of progesterone decline.

Dietary Apigenin Exerts Immune-Regulatory Activity in Vivo by Reducing NF-κB Activity, Halting Leukocyte Infiltration and Restoring Normal Metabolic Function

The increasing prevalence of inflammatory diseases and the adverse effects associated with the long-term use of current anti-inflammatory therapies prompt the identification of alternative approaches to reestablish immune balance. Apigenin, an abundant dietary flavonoid, is emerging as a potential regulator of inflammation. Here, we show that apigenin has immune-regulatory activity in vivo. Apigenin conferred survival to mice treated with a lethal dose of Lipopolysaccharide (LPS) restoring normal cardiac function and heart mitochondrial Complex I activity. Despite the adverse effects associated with high levels of splenocyte apoptosis in septic models, apigenin had no effect on reducing cell death. However, we found that apigenin decreased LPS-induced apoptosis in lungs, infiltration of inflammatory cells and chemotactic factors’ accumulation, re-establishing normal lung architecture. Using NF-κB luciferase transgenic mice, we found that apigenin effectively modulated NF-κB activity in the lungs, suggesting the ability of dietary compounds to exert immune-regulatory activity in an organ-specific manner. Collectively, these findings provide novel insights into the underlying immune-regulatory mechanisms of dietary nutraceuticals in vivo.

Dihydrotestosterone influenced numbers of healthy follicles and follicular amounts of LH receptor mRNA during the follicular phase of the estrous cycle in gilts

The present experiments were conducted to determine androgenic effects on numbers, health, and amounts of gonadotropin receptor mRNA in late developing follicles of gilts. Gilts (n=5 per group) received daily injections of one of the following treatments on days 13-16 or days 13-18 of the estrous cycle: corn oil, 5alpha-dihydrotestosterone (DHT, 10 mg), flutamide (1.5 g, an androgen receptor inhibitor), DHT (10 mg) plus flutamide (1.5 g), testosterone (10 mg), and testosterone (10 mg) plus flutamide (1.5 g). Ovarian follicles > or =5 mm in diameter were evaluated on day 17 or 19, 24 h after receiving the last treatment dose. Follicles were classified as healthy (H), moderately atretic (MA), or very atretic (VA). Treatment with DHT increased (P<0.05) the numbers of H follicles relative to control gilts on days 17 and 19. DHT administration from days 13 to 16 diminished (P<0.05) the amounts of LH receptor (LHR) mRNA in H follicles from day 17 (relative amounts: 1.45+/-0.33 and 2.72+/-0.33 for DHT- and vehicle-treated gilts respectively). The effects of DHT on numbers of H follicles and LHR mRNA were not observed in gilts receiving DHT plus flutamide. Androgens did not influence numbers of MA, VA, and total follicles, or follicular estradiol-17beta concentrations and amounts of FSHR mRNA. Treating gilts with DHT during follicular recruitment and selection did not induce changes in the numbers of total follicles > or =5 mm, but rather increased the numbers of healthy follicles in this follicular population in association with decreased amounts of LHR mRNA.