Modulation of PGE2-induced EP4 expression on snail signaling and the impact on epithelial-mesenchymal transition: significance of EP4 antagonism.

BACKGROUND: Although significant accumulation of prostaglandin E(2) (PGE(2)) in the human prostate cancer tissues has been reported, there is lack of substantial evidence regarding the key role of PGE(2)-induced E-prostanoid-4 receptor (EP4) on Snail, a master regulator of epithelial mesenchymal transition (EMT). In this study, we investigated a novel connection between PGE(2)-induced EP4 and Snail (encodes DNA binding zinc finger protein that acts as transcriptional repressor) signaling in prostate cancer. MATERIALS AND METHODS: To investigate the key role of serum PGE(2), EP4, p-Akt and Snail in prostate cancer progression, we used prostate-specific phosphatase and tensin homolog (PTEN)-knockout (PTEN-KO) mice of different age groups from 4 to 28 weeks. To determine the EP4-specific interaction with Snail in prostate cancer, we used cell-based assays, including siRNA knockdown, and treatment with EP4 antagonist. RESULTS: An interaction between EP4 with Snail was evident in prostate-specific PTEN-KO mice that showed an elevated level of PGE(2) in the serum and of EP4, p-Akt and Snail in the tissues. Prostate cancer cells transfected with EP4-siRNA and treatments with EP4 antagonist suggest a link between EP4, and Snail activation, potentially via p-Akt. Cells treated with EP4 antagonist exhibited a significant decrease in Snail, mesenchymal markers and cell migration, and cell cycle arrest with a gain in E-cadherin levels. CONCLUSION: Our findings provide key evidence that support there being a role of PGE(2)/EP4/p-Akt in Snail signaling and conferring cell survival advantage. Cancer progression via EMT can be reversed by an EP4 antagonist in this model of prostate cancer.

Relaxin and progesterone during pregnancy and the post-partum period in association with live and stillborn calves in bottlenose dolphins (Tursiops truncatus).

The objectives of this study were to validate a relaxin and progesterone RIA for use in bottlenose dolphins, and quantify and characterize both hormones in extracts of placental tissue and serum collected during pregnancy and the post-partum period, and compare the results between dolphins with live and stillborn calves. In Experiment 1, validation of a heterologous relaxin and progesterone RIA involved specific displacement of antibody-bound radiolabeled human relaxin or progesterone in response to increasing volumes of pooled pregnant dolphin serum and amounts of respective hormone standards added to a fixed volume of serum. The displacement curves were considered parallel and additive relative to respective standard curves. In Experiment 2, immunoreactive relaxin and progesterone were detected in placental extracts and, in corresponding serum samples, concentrations of both hormones were higher during the pre-partum than post-partum periods. Circulatory concentrations of progesterone decreased (P < 0.05) from relatively high concentrations during early and mid-pregnancy to intermediate concentrations by late pregnancy (month effect, P < 0.0001) in dolphins with live births, whereas, in dolphins with stillbirths, the decrease in progesterone began earlier (month-by-birth status interaction, P < 0.007); mean concentrations were lower at mid- (37%, P < 0.06) and late (25%) pregnancy. Temporally, relaxin increased (P < 0.05) progressively from relatively low concentrations during early pregnancy to high concentrations during late pregnancy (month effect, P < 0.0001) and was not different between birth statuses (birth status effect, P = 0.76; month-by-birth status interaction, P = 0.17). Even though the interaction did not reach significance, mean relaxin concentrations were 42%, 29%, and 34% lower at early, mid-, and late pregnancy, respectively, in dolphins with stillbirths than in those with live births. In conclusion, the pregnancy-specific increase in serum concentrations of relaxin and lower concentrations of both relaxin and progesterone in association with stillbirths suggest the potential for relaxin to be used diagnostically to determine pregnancy status, and one or both hormones to be used to assess placental function, and, perhaps, fetal well-being in bottlenose dolphins and other cetaceans.

Characterization and biological activity of relaxin in porcine milk.

A lactocrine mechanism for delivery of maternally derived relaxin (RLX) into the neonatal circulation as a consequence of nursing was proposed for the pig. Immunoreactive RLX was detected in colostrum and in the serum of newborn pigs only if they were allowed to nurse. Milk-borne RLX concentrations are highest during early lactation (9-19  ng/ml), declining to <2  ng/ml by postnatal day 14. Whether milk-borne RLX is bioactive is unknown. Evidence that RLX concentrations in milk are higher than in maternal circulation in several species suggests the mammary gland as a site of local RLX production. It is unknown whether the porcine mammary gland is a source of RLX. Therefore, objectives were to evaluate RLX bioactivity in porcine milk during the first 2 weeks of lactation, identify the form of RLX in porcine milk, and determine whether mammary tissue from early lactation is a source of milk-borne RLX. Milk RLX bioactivity was determined using an in vitro bioassay in which cAMP production by human embryonic kidney (HEK293T) cells transfected with the human RLX receptor (RXFP1) was measured. RLX bioactivity was highest at lactation day (LD) 0, decreasing to undetectable levels by LD 4. Immunoblot analysis of milk proteins revealed an 18  kDa band, indicating proRLX as the primary form of RLX in porcine milk. ProRLX protein and transcripts were detected in porcine mammary tissue on LD 0 and 7. Results support the lactocrine hypothesis by defining the nature and a potential source for bioactive proRLX in porcine colostrum/milk.

Selenomethionine and alpha-tocopherol do not inhibit prostate carcinogenesis in the testosterone plus estradiol-treated NBL rat model.

Previous studies with selenium and/or vitamin E in prostate carcinogenesis animal models have been negative, but these models may not involve oxidative stress mechanisms. In this study, we examined the potential of selenomethionine and alpha-tocopherol to modulate prostate cancer development in the testosterone plus estradiol-treated NBL rat, a model that does involve sex hormone-induced oxidative stress mechanisms and prostatic inflammation. One week following the implantation with hormone-filled Silastic implants, rats were fed diets containing l-selenomethionine (1.5 or 3.0 mg/kg), DL-alpha-tocopherol acetate (2,000 or 4,000 mg/kg), or a natural ingredient control diet (NIH-07). The development of prostate carcinomas was not affected by dietary treatment with either agent. Food intake, body weight, and mortality were also not affected. The high dose of selenomethionine reduced the severity of epithelial dysplasia in the lateral prostate that was not associated with inflammation, and alpha-tocopherol reduced in a dose-related fashion the incidence of marked inflammation and marked epithelial dysplasia in the lateral prostate, regardless of whether these lesions were associated with inflammation. alpha-Tocopherol significantly increased the incidence of adenocarcinomas of the mammary glands at both dietary concentrations. Collectively, our findings suggest that selenomethionine and alpha-tocopherol supplementation does not prevent prostate cancer in rats fed diets with nutritionally adequate levels of selenium and vitamin E. Importantly, the results of the current animal studies and those reported previously were fully predictive of the outcome of the Selenium and Vitamin E Cancer Prevention Trial.

Hormonal changes accompanying cigarette smoke-induced preterm births in a mouse model.

Epidemiologic evidence indicates that maternal smoking increases the risk of preterm birth. While a number of plausible mechanisms for early delivery have been offered, the role of gestational hormones in this smoke-induced outcome is uncertain. Thus, a toxicologic study was performed to examine the effects and underlying hormonal mechanisms of mainstream cigarette smoke (MCS) exposure on gestational duration. Pregnant B6C3F1 mice were exposed by inhalation to MCS for 5 days/week (4 hrs/day) from Gestational Day (GD) 4 to parturition. Smoke-induced effects on gestational length, interpubic ligament length, maternal hormone secretion patterns (estradiol-17beta, progesterone, prolactin, and relaxin), body weight gain, postimplantation loss, litter size, and offspring sex ratio were examined. Dams exposed to MCS at a concentration equivalent to smoking less than one pack of cigarettes/day (carbon monoxide = 25 parts per million, total suspended particulates = 16 mg/m3) demonstrated a significant (P < 0.05) shortening of gestational duration (compared with pregnant, air-exposed mice). In addition, MCS-exposed mice sacrificed on GD 18 had significantly (P < 0.05) increased interpubic ligament length, elevated serum estrogen levels, and a reduced progesterone to estradiol-17beta ratio (compared with air-exposed controls); levels of progesterone and prolactin were only modestly decreased and increased, respectively, in the MCS-exposed mice. Smoke exposure had no significant effects on maternal relaxin levels, body weight gain, postimplantation loss, litter size, or sex ratio. Results of this study demonstrate that inhalation exposure of pregnant mice to a low dose of MCS shortens gestation and alters hormone secretory patterns, which are important for maintaining pregnancy and inducing parturition. These findings support the view that pregnant women who smoke (even modestly) may be at increased risk for preterm birth, and that early delivery may be related (at least partly) to MCS-induced.

The parity-related protection against breast cancer is compromised by cigarette smoke during rat pregnancy: observations on tumorigenesis and immunological defenses of the neonate.

Early pregnancy is a powerful negative risk factor for breast cancer (BCa) in women. Pregnancy also protects rats against induction of BCa by carcinogens such as N-methyl-N-nitrosourea (MNU), making the parous rat a useful model for studying this phenomenon. Smoking during early pregnancy may lead to an increased risk of BCa in later life, possibly attributable to carcinogens in cigarette smoke (CS), or to reversal of the parity-related protection against BCa. To investigate these possibilities, 50-day-old timed first-pregnancy rats were exposed to standardized mainstream CS (particle concentration = 50 mg/m3) or to filtered air (FA) 4 h/day, Day 2-20 of gestation. Age-matched virgin rats were similarly exposed to CS or FA. At age 100 days, the CS or FA-exposed, parous and virgin rats were injected s.c. with MNU (50 mg/kg body wt), or with MNU vehicle. Mammary tumors (MTs) first appeared in virgin rats 9 weeks post-MNU injection. While no MTs were detected in FA-exposed parous rats until 18 weeks post-MNU, MTs appeared in the CS-exposed parous rats as early as 10 wks (P < 0.02). As no MTs developed in CS-exposed rats not injected with MNU, CS did not act as a direct mammary carcinogen. Serum prolactin concentration on Day 19 of pregnancy in CS-exposed dams was reduced by 50% compared with FA-exposed dams (P < 0.005). CS exposure during a pregnancy may thus 'deprotect' rats, enhancing their vulnerability to MNU-induced BCa. Prenatal CS exposure had no detectable effect on the immune responses of the pups examined at 3, 8 or 19 weeks of age. However, prolactin concentration in stomach contents (milk) of 3-day-old pups suckled by CS-exposed dams was decreased when compared with that of FA-exposed dams (P < 0.032). As milk-borne prolactin modulates development of the central nervous and immune systems of neonatal rats, CS exposure of the dams could adversely affect later maturation of these systems by reducing milk prolactin.