microM E2 Research Articles

Environmental endocrine disruptors promote invasion and metastasis of SK-N-SH human neuroblastoma cells

Neuroblastoma (NB) is the most common pediatric extracranial cancer. Metastasis is the main cause of mortality in NB patients. Currently, little is known about the risk factors and their mechanisms that cause metastasis. Environmental endocrine disruptors (EED) are recently identified risk factors associated with various human diseases including malignant tumors. Our previous studies have implicated the role of di(2-ethylhexyl) phthalate (DEHP) and bisphenol A (BPA), two of the most common EED, in neuroblastoma cell proliferation. Here, we further investigated the effects of DEHP, BPA as well as 17beta-estradiol (E2) on the invasion and metastasis of human neuroblastoma SK-N-SH cells in vitro. SK-N-SH cells expressed estrogen receptor (ER)-beta, matrix metalloproteinases-2 (MMP-2), MMP-9 and tissue inhibitor of matrix metalloproteinase-2 (TIMP-2) at readily detectable levels. 50 microM DEHP, 0.1 microM BPA and 10 microM E2 exposure all resulted in enhanced motility and invasiveness of SK-N-SH cells (P<0.001), elevated expression of MMP-2 and MMP-9, and decreased expression of TIMP-2 (P<0.01). Furthermore, phosphorylation of Akt (Ser473) was also induced following the exposure (P<0.01). Importantly, both ER antagonist ICI182,780 and phosphoinositide 3-kinase (PI3K) specific inhibitor LY294002 significantly inhibited the DEHP, BPA, or E2-induced cell migration and invasion, as well as the disregulation of MMP-2, MMP-9 and TIMP-2 expression. ICI182,780 may have worked through abolishing Akt (Ser473) phosphorylation. In conclusion, DEHP, BPA, and E2 potently promote invasion and metastasis of neuroblastoma cells through overexpression of MMP-2 and MMP-9 as well as downregulation of TIMP-2. ER-dependent pathway and PI3K/Akt pathway are involved, which may become potential therapeutic targets for neuroblastoma treatment.

Effects of 17β-Estradiol on Neuronal Cell Excitability and Neurotransmission in the Suprachiasmatic Nucleus of Rat

17beta-Estradiol receptors have been found in several brain nuclei including the suprachiasmatic nucleus (SCN) of mammalian species. The SCN is believed to act as brain clock regulating circadian and circannual biological rhythms, such as body temperature, sleep, and mood. Here, we examined whether 17beta-estradiol (E2) could affect cell excitability and synaptic transmission in the SCN. Bath application of E2 (0.03-3 microM) increased the spontaneous firing frequency and depolarized cell membrane of the SCN neurons significantly. Furthermore, E2 (0.03-3 microM) increased (by about 25-150% of control) frequency of the miniature excitatory postsynaptic currents. Amplitude of the evoked excitatory postsynaptic currents was enhanced (by about 32% of control) after exposure to 1 microM E2. The paired-pulse ratio was reduced by E2. These effects were prevented by the estrogen receptor antagonist, ICI 182780. Exposure to the biologically inactive 17alpha-estradiol did not cause any significant changes in the parameters mentioned above. These findings are in favor of an implication of estrogen in modulation of neuronal activity in SCN and possibly regulating circadian rhythms.

17β-Estradiol Potentiates the Cardiac Cystic Fibrosis Transmembrane Conductance Regulator Chloride Current in Guinea-pig Ventricular Myocytes

There is a well-characterized membrane chloride current (ICl,cAMP) in the heart that can be activated by beta-adrenergic agonists and is due to expression of the cardiac isoform of the epithelial cystic fibrosis transmembrane conductance regulator (CFTR). We have investigated whether 17beta-estradiol (E2) modulates ICl,cAMP in single ventricular myocytes. Under whole-cell tight-seal voltage-clamp conditions, ICl,cAMP was evoked by exposing cells to 20 nM isoprenaline. On the addition of 30 microM E2, membrane slope conductance, measured at potentials near 0 mV, increased over that induced by isoprenaline alone by 2.46 +/- 0.16 (p < 0.001). The effects of E2 were concentration-dependent and described by a Hill Plot with an EC50 of 8.2 microM and a Hill coefficient of 1.63. The application of membrane-impermeant E2 conjugated to bovine serum albumin (E2-BSA) potentiated isoprenaline-evoked ICl,cAMP by approximately the same degree as that for the equivalent level of free E2. Cell surface binding was observed with confocal microscopy by using BSA-FITC tagged E2. This binding was inhibited by nonlabeled, nonconjugate E2, the specific E2 antagonist ICl 182,780, and incubation of E2coBSA with a specific anti-E2 antibody (E2885). ICl 182,780 (100 microM) significantly reduced the increase in ICl,cAMP evoked by 10 microM E2 to 1.46 +/- 0.10 (p < 0.02). The preincubation of myocytes with the NOS inhibitor N-omega-nitro-arginine (L-NNA, 1 mM) reduced the potentiation of ICl,cAMP by 30 microM E2, to 1.93 +/- 0.06 (p < 0.02), and for 10 microM E2, to 1.32 +/- 0.05 (p < 0.002). E2 also increased ICl,cAMP evoked by bath application of 0.5 microM Forskolin. These experiments demonstrate that, under our experimental conditions, E2 dramatically increases ICl,cAMP in ventricular myocytes by mechanisms involving a contribution by NOS, but that can be only partially accounted for through binding to classical plasma membrane estrogen receptor sites. This potentiation of ICl,cAMP by E2 may play a significant role in the observed clinical actions of E2 on the incidence of cardiac arrhythmias and hypertrophy.

Activated Function of the Pyruvate Dehydrogenase Phosphatase through Ca2+-facilitated Binding to the Inner Lipoyl Domain of the Dihydrolipoyl Acetyltransferase

Micromolar Ca2+ facilitates approximately 10-fold enhancement of pyruvate dehydrogenase phosphatase (PDP) activity by aiding the association of PDP with the dihydrolipoyl acetyltransferase (E2) component. Connected by linker regions, E2 consists of two lipoyl domains, the NH2-lipoyl domain (L1) and the interior lipoyl domain (L2), and a pyruvate dehydrogenase component binding domain surrounding a 60-mer inner core. Using recombinant constructs of L1 or L2, E2-enhanced PDP activity was markedly decreased by L2 but not by L1, effectively competing with intact E2 in Ca2+-dependent binding of PDP (half-maximal reduction at 2.0 microM L2 versus 6.7 microM E2 subunit). Using L2 fused to glutathione S-transferase resulted in direct Ca2+-dependent binding of PDP to L2 (Kd, approximately 1.7 microM L2). Affinity-bound glutathione S-transferase-L2 was used to purify PDP to homogeneity by selective binding and elution by Ca2+ chelation. The large activity enhancement of PDP by E2 was eliminated by enzymatic removal of lipoates from E2 and restored by their enzymatic reintroduction. The critical role of the L2 lipoate is not in binding of PDP to E2, since PDP was still bound by delipoylated L2, and delipoylated L2 inhibited E2-enhanced PDP activity, although lipoylated L2 was more effective in each of these tests. Thus, pyruvate dehydrogenase complex activity is increased by enhanced availability of PDP to its E2-bound, phosphorylated pyruvate dehydrogenase substrate as a consequence of the Ca2+-facilitated interchange of PDP among the mobile L2 domains and an essential (undetermined) step engaging the L2 lipoate.

Effect of estradiol and ethynylestradiol on microtubule distribution in Chinese hamster V79 cells.

The effects of estradiol (E2) and ethynylestradiol (EE2) on the chromosome number and cellular microtubule architecture of Chinese hamster V79 cells were studied using fluorescent anti-tubulin antibody. Treatment with 20 microM E2 for 48 h induced only a small amount of tetraploid cells, but the normal microtubule network was disrupted completely by only 3 h of treatment. This data reveals that E2 has higher microtubule-disruptive activity than diethylstilbestrol in V79 cells.

Proliferative inhibition of human breast carcinoma cells by high concentration estradiol does not alter radiosensitivity

The proliferation of ER+ malignant mammary epithelial cells (MMEC), MCF-7 and T-47D, was markedly inhibited by 10 microM 17 beta-estradiol (E2), while the ER- line, MDA-MB-231, was minimally affected. This concentration of E2 did not alter the proportion of non-viable cells or the plating efficiency. The decrease in proliferation was not associated with any consistent alteration in the cell kinetic profile between lines, though there was clearly no dominant cell cycle re-assortment after a two day incubation. Colony forming ability after exposure to ionizing irradiation was compared for proliferating and confluent MMEC to cells incubated in 10 microM E2 for two days. The presence of E2 resulted in no significant differences for any of the linear quadratic curve fitting parameters or for mean inactivation doses for both subconfluent and confluent cultures. Further, 10 microM E2 had no effect on the ability of cells to recover from split radiation fractions. There were no differences in the amount of DNA single strand break induction with E2, but there was a significant shortening in the repair halftime with E2 for the ER- MDA-MB-231 cells. Though high concentrations of E2 can markedly inhibit the proliferation of ER+ MMEC, this does not alter sensitivity to ionizing irradiation.

In vitro synthesis of prostaglandin E and F2 alpha by pig endometrium in the presence of estradiol, catechol estrogen and ascorbic acid.

These experiments were undertaken to determine the potential for estradiol-17 beta (E2), 2-hydroxyestradiol-17 beta (2-OH-E2) and 4-hydroxyestradiol-17 beta (4-OH-E2) to regulate prostaglandin (PG) E and F2 alpha synthesis by pig endometrium. Endometrium was collected from pigs on d 10 of pregnancy and incubated (15 to 20 mg/well) for three 2-h periods in 2 ml of medium in 24-well culture plates. At the end of each period, the medium was removed and frozen. Later media were thawed and assayed for PGE and PGF2 alpha. During Periods 2 and 3, the medium contained 0, 25, 50, 100 or 150 microM 2-OH-E2 (Exp. 1); 0, 25 or 50 microM 4-OH-E2 (Exp. 2); or 0, 25 or 50 microM E2 (Exp. 3). Each experiment was a factorial with 2-OH-E2, 4-OH-E2 or E2 as one main effect and 0 or 1 mM ascorbate as a second main effect. Ascorbate decreased (P less than .01) PGE and PGF2 alpha release in all experiments. Two-hydroxyestradiol-17 beta decreased (P less than .01) PGE and PGF2 alpha release into the medium during Periods 2 and 3 in a dose-dependent manner (Exp. 1). In Exp. 2, 4-OH-E2 decreased (P less than .07) endometrial release of PGE and PGF2 alpha in Periods 2 and 3 and increased (P less than .01) the PGE:PGF2 alpha in Period 3. In Exp. 3, E2 decreased release of PGE during Period 3 and PGF2 alpha release during Period 2. The PGE:PGF2 alpha was not altered by E2.(ABSTRACT TRUNCATED AT 250 WORDS)

The effect of estrogen on androstenedione production by rat placental cells in vitro.

We have recently provided evidence from studies conducted in vivo that the ovary, particularly by means of estrogen, regulates placental androstenedione (delta 4A) production during the second half of rat pregnancy. In the present study, an incubation system of dispersed rat placental cells was established to determine if estrogen acts directly on the placenta to regulate delta 4A production. Placentas were obtained on Days 14-15 of rat gestation and dispersed in Hanks' Balanced Salt Solution containing 0.1% collagenase, 0.1% hyaluronidase, 0.01% DNase, and 1% fetal calf serum. Placental cells were incubated in Medium 199 for 16 h at 37 degrees C. A time-dependent increase (r = 0.96, p less than 0.05) in the release of delta 4A occurred over the 16-h incubation period. Mean +/- SE formation of the steroid intermediate progesterone (P4) and product delta 4A was 1.17 +/- 0.78 and 1.18 +/- 0.22 ng per 10(7) cells respectively. The addition of 1-10 microM diethylstilbestrol (DES) decreased (p less than 0.05-0.01) delta 4A production, and had no significant effect on P4 or pregnenolone (P5) formation. The percent decrease in delta 4A production was 14.2 +/- 12.9, 30.9 +/- 2.3, and 55.0 +/- 4.4 with 1, 5, and 10 microM DES, respectively. Treatment of placental cells with estradiol (E2) also resulted in a decrease (p less than 0.01) in delta 4A production with no effect on P4 formation. The percent inhibition of delta 4A production was 34.2 +/- 11.1 and 77.3 +/- 5.2 with the addition of 1 microM and 10 microM E2, respectively. E2 (10 microM) produced a concomitant threefold increase (p less than 0.01) in P5 formation.(ABSTRACT TRUNCATED AT 250 WORDS)