Identificación de Centros Funcionales

A number of structurally-diverse estrogenic compounds were used to test their abilities to activate kinases in MCF-7 cells (Fig. 53). Cells were transfected with GAL4-Elk- 1/ERα, treated with 10 nM E2 and DES and various concentrations (20-75 uM) of other estrogenic compounds. All compounds, with the exception of resveratrol, induced luciferase activity, and thereby activated MAPK activity (Fig. 54)

.

Fig. 53. Estrogenic compounds tested for their activation of kinase pathways in MCF-7 cells.

GAL4-Elk-1

Relative Luciferase Activity

0 20 40 60 D 10 nM E2 25 uM HPTE 25 uM Octylphenol 25 uM Nonylphenol 50 uM Endosulfan 75 uM Resveratrol 75 uM BPA 20 uM Kepone 25 uM HO-PCB-Cl4 10 nM DES

*

*

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*

*

*

*

*

*

*

*

Fig. 54. Activation of GAL4-Elk-1 by estrogenic compounds. MCF-7 cells were transfected with 1ug GAL4-Luc, 0.5 ug ER, and 1 ug GAL4-Elk-l. Transfected cells were treated with D (DMSO), E2, or the estrogenic compounds for 24 hr. Luciferase activities were determined as described in the Materials and Methods. Significant induction (p<0.05) compared to DMSO control is indicated by an asterisk.

Results from Western blot analysis for phospho-ERK confirmed results of transient transfection assays. With the exception of resveratrol, E2 and other estrogenic compounds induced phosphorylation of ERK within 10 min after treatment (Fig. 55). Inhibition studies with PD98059 indicated that E2 and two other xenoestrogenic compounds, HPTE and nonylphenol, also induced phosphorylation of ERK (Fig. 56).

p-ERK

GAPDH

Fig. 55. Effects of estrogenic compounds on the phosphorylation of ERK. MCF-7 cells were treated for 10 min with D (DMSO), E2, HPTE, Oct (p-t-octylphenol), Non (nonylphenol), Endo (endosulfan), Res (resveratrol), BPA, Kep (Kepone), PCB (HO-PCB-Cl4) and DES. Whole cell lysates were prepared and Western

blots were performed as described in the Materials and Methods. GAPDH was used as a loading control.

D E2 E2+PD D HPTE HPTE+PD D Non Non+PD

p-ERK GAPDH Fig. 56. Inhibition of ERK phosphorylation by PD98059 (PD). MCF-7 cells were pretreated with 30 uM PD98059 for 2 hr and then treated with D, E2, HPTE or Non in the presence or absence of PD. Whole cell lysates were prepared and Western blots were performed as described in the Materials and Methods. GAPDH was used as a loading control.

GAL4-SRF

Relative Luciferase Activity

0 20 40 6 D 10 nM E2 25 uM HPTE 25 uM Octylphenol 25 uM Nonylphenol 50 uM Endosulfan 75 uM Resveratrol 75 uM BPA 20 uM Kepone 25 uM HO-PCB-Cl4 10 nM DES

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Fig. 57. Activation of GAL4-SRF by estrogenic compounds. MCF-7 cells were transfected with 1ug GAL4-Luc, 0.5 ug ER, and 0.1 ug GAL4-SRF. Transfected cells were treated with D (DMSO), E2, or the estrogenic compounds for 24 hr. Luciferase activities were determined as described in the Materials and Methods. Significant induction (p<0.05) compared to DMSO control is indicated by an asterisk.

Estrogen-induced PI3K plays a major role in proliferation of MCF-7 cells (Castoria et al., 2001). Results in Figure 57 showed that E2, DES and seven xenoestrogens all induced PI3K activity whereas induction was not observable for resveratrol. Western analysis for phosphorylation of AKT, a downstream target of PI3K, confirmed this observation (Fig. 58). The relative potencies of these compounds varied among the transient transfection and Western blot assays, possibly due to the fact that transient transfection assay evaluates long term (24 hr) effects whereas short term (10 min) effects are observed in the immuno blots. The results in Figure 59 confirmed that the induction of AKT phosphorylation by E2, nonylphenol and HPTE is inhibited by the PI3K inhibitor LY294002.

p-AKT

GAPDH

Fig. 58. Effects of estrogenic compounds on phosphorylation of AKT. MCF-7 cells were treated for 10 min with D (DMSO), E2, HPTE, Oct (p-t-octylphenol), Non (nonylphenol), Endo (endosulfan), Res (resveratrol), BPA, Kep (Kepone), PCB (HO-PCB-Cl4) and DES. Whole cell lysates were prepared and Western

blots were performed as described in the Materials and Methods. GAPDH was used as a loading control.

p-AKT GAPDH

D E2 E2+LY D HPTE HPTE+LY D Non Non+LY

Fig. 59. Inhibition of AKT phosphorylation by LY294002 (LY). MCF-7 cells were pretreated with 25 uM LY for 2 hr and then treated with D, E2, HPTE or Non in the presence or absence of LY. Whole cell lysates were prepared and Western blots were performed as described in the Materials and Methods. GAPDH was used as a loading control.

GAL4-CREB

Relative Luciferase Activity

0

4

8

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Fig. 60. Activation of GAL4-CREB by estrogenic compounds. MCF-7 cells were transfected with 1ug GAL4-Luc, 0.5 ug ER, and 0.1 ug GAL4-CREB. Transfected cells were treated with D (DMSO), E2, or the estrogenic compounds for 24 hr. Luciferase activities were determined as described in the Materials and Methods. Significant induction (p<0.05) compared to DMSO control is indicated by an asterisk.

Activation of GAL4-CREB is observed in MCF-7 cells treated with E2, DES, HPTE, BPA, octylphenol, nonylphenol and endosulfan, but not kepone, resveratrol and HO- PCB-Cl4. These results (Fig. 60) differentiated kepone and HO-PCB-Cl4 from other

xenoestrogens. Transactivation studies suggested that E2 primarily activates CREB through PKC pathways in MCF-7 cells (Fig. 51), and this was further investigated by analyzing induction of CREB phosphorylation by PKC in the presence or the absence of PKC inhibitor Ro-31-8425 and PKA inhibitor H89. Minor (E2/HPTE) or no detectable inhibition (nonylphenol) of CREB phosphorylation were observed in cells cotreated with H89. Significant inhibition was observed after treatment with E2, HPTE and nonylphenol in cells cotreated with Ro-31-8425 (Fig. 61). These results confirmed that E2 and some xenoestrogens activate PKC in MCF-7 cells.

p-CREB GAPDH

D E2 E2 E2 D HPTE HPTE HPTE D Non Non Non

+ +

H89 Ro H89 Ro+ + H89 Ro+ +

Fig. 61. Effects of estrogenic compounds on the phosphorylation of CREB. MCF- 7 cells were pretreated with 10 uM H89 or 5 uM Ro-31-8425 (Ro) for 2 hr and then treated with D, E, HPTE or Non (nonylphenol) in the presence or absence of inhibitors for 10 min. Whole cell lysates were prepared and Western blots were performed as described in the Materials and Methods. GAPDH was used as a loading control.

GAL4-p65

Relative Luciferase Activity

0 20 40 D 10 nM E2 25 uM HPTE 25 uM Octylphenol 25 uM Nonylphenol 50 uM Endosulfan 75 uM Resveratrol 75 uM BPA 20 uM Kepone 25 uM HO-PCB-Cl4 10 nM DES

*

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Fig. 62. Activation of GAL4-p65 by estrogenic compounds. MCF-7 cells were transfected with 1ug GAL4-Luc, 0.5 ug ER, and 0.1 ug GAL4-p65. Transfected cells were treated with D (DMSO), E2, or the estrogenic compounds for 24 hr. Luciferase activities were determined as described in the Materials and Methods. Significant induction (p<0.05) compared to DMSO control is indicated by an asterisk.

Of all the compounds tested, only E2 and DES induced transactivation in MCF-7 cells transfected with GAL4-p65/ERα (Fig. 62). However, preliminary studies showed that in cells transfected with GAL4-p65, HO-PCB-Cl4 inhibited the E2-induced transactivation,

suggesting that this compound may be a CaMKIV inhibitor. This inhibitory effect was further investigated in MCF-7 cells transfected with GAL4-p65/ERα and treated with E2 alone or in combination with different concentrations of HO-PCB-Cl4. The results

showed that HO-PCB-Cl4 significantly inhibited CaMKIV-dependent activation of GAL4-

p65 by E2 in a dose-dependent manner (Fig. 63). Antibodies for detecting phospho- CaMKIV are not available. Therefore, the inhibitory effects of HO-PCB-Cl4 were

investigated using CaMKIV-dependent induction of p53 by E2 as a model (Qin et al., 2002). The results (Fig. 64) demonstrated that E2-dependent inductions of p53 protein levels in MCF-7 cells was inhibited by the antiestrogen ICI 182,780 and by both KN93 and HO-PCB-Cl4, confirming that HO-PCB-Cl4 is a CaMKIV inhibitor.

0 2 D E2 25 uM 10 uM 5 uM 1 uM 0.75 uM 0.5 uM

Relative Luciferase Activity

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Fig. 63. Inhibition of transactivation of GAL4-p65 by HO-PCB-Cl4. MCF-7 cells

were transfected with 1ug GAL4-Luc, 0.5 ug ER and 0.1ug GAL4-p65. Cells were treated with D, E2 or E2 plus various concentrations of HO-PCB-Cl4 for 24 hr.

Luciferase activities were determined described in the Materials and Methods. Significant induction (p<0.05) compared to DMSO control is indicated by an asterisk.

D E2 E2+ICI E2+KN E2+PCB

p53 GAPDH

Fig. 64. Inhibition of hormone-induced p53 protein expression by ICI 182, 780, KN93 and HO-PCB-Cl4. MCF-7 cells were treated with DMSO (D), E2, or E2 plus

ICI 182,780 (ICI), KN93 (KN) and HO-PCB-Cl4 (PCB) for 6 hr. Whole cell lysates

were prepared and Western blots were performed as described in the Materials

and Methods. GAPDH was used a loading control.

To evaluate the role of ER in the nongenomic actions of estrogenic compounds, we chose E2 and two compounds HPTE and nonylphenol to test whether their activation of MAPK and PI3K is sensitive to antiestrogen inhibition. The phosphorylation of AKT through E2, HPTE and nonylphenol were inhibited by ICI 182, 780, suggesting that activation of PI3K by these three chemicals is ER-dependent. E2-induced phosphorylation of ERK is also inhibited by ICI 182,782, however, HPTE- and nonylphenol-induced phosphorylation of ERK were partially (HPTE) or completely (nonylphenol) resistant to the inhibitory effects of ICI 182,780 (Fig. 65). These results indicate that some nongenomic actions of xenoestrogen may not be mediated by ER.

D E E+I D H H+I D N N+I

p-ERK p-AKT

GAPDH

Fig. 65. Effects of the antiestrogen ICI 182,780 on MAPK and PI3K activation by estrogenic compounds. MCF-7 cells were pretreated with ICI 182,780 (I) for 6 hr and then treated with DMSO (D), E2 (E), HPTE (H) and nonylphenol (N) for 10 min. Whole cell lysates were prepared and Western blots were performed as described in the Materials and Methods. GAPDH was used as a loading control.

CHAPTER IV

DISCUSSION AND SUMMARY