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Estrogen Receptor β Agonists Differentially Affect the Growth of Human Melanoma Cell Lines.

Marzagalli M, Casati L, Moretti RM, Montagnani Marelli M, Limonta P - PLoS ONE (2015)

Bottom Line: Moreover, the antiproliferative activity of ERβ agonists was associated with an altered expression of G1-S transition-related proteins.In these cells, global DNA was found to be hypomethylated when compared to normal melanocytes; this DNA hypomethylation status was reverted by ERβ activation.ERβ agonists also decreased the proliferation of WM115 (BRAF V600D-mutant) cells, while they failed to reduce the growth of A375 and WM1552 (BRAF V600E-mutant) cells.

View Article: PubMed Central - PubMed

Affiliation: Department of Pharmacological and Biomolecular Sciences, Università degli Studi di Milano, Milano, Italy.

ABSTRACT

Background: Cutaneous melanoma is an aggressive malignancy; its incidence is increasing worldwide and its prognosis remains poor. Clinical observations indicate that estrogen receptor β (ERβ) is expressed in melanoma tissues and its expression decreases with tumor progression, suggesting its tumor suppressive function. These experiments were performed to investigate the effects of ERβ activation on melanoma cell growth.

Methods and results: Protein expression was analyzed by Western blot and immunofluorescence assays. Cell proliferation was assessed by counting the cells by hemocytometer. ERβ transcriptional activity was evaluated by gene reporter assay. Global DNA methylation was analyzed by restriction enzyme assay and ERβ isoforms were identified by qRT-PCR. We demonstrated that ERβ is expressed in a panel of human melanoma cell lines (BLM, WM115, A375, WM1552). In BLM (NRAS-mutant) cells, ERβ agonists significantly and specifically inhibited cell proliferation. ERβ activation triggered its cytoplasmic-to-nuclear translocation and transcriptional activity. Moreover, the antiproliferative activity of ERβ agonists was associated with an altered expression of G1-S transition-related proteins. In these cells, global DNA was found to be hypomethylated when compared to normal melanocytes; this DNA hypomethylation status was reverted by ERβ activation. ERβ agonists also decreased the proliferation of WM115 (BRAF V600D-mutant) cells, while they failed to reduce the growth of A375 and WM1552 (BRAF V600E-mutant) cells. Finally, we could observe that ERβ isoforms are expressed at different levels in the various cell lines. Specific oncogenic mutations or differential expression of receptor isoforms might be responsible for the different responses of cell lines to ERβ agonists.

Conclusions: Our results demonstrate that ERβ is expressed in melanoma cell lines and that ERβ agonists differentially regulate the proliferation of these cells. These data confirm the notion that melanoma is a heterogeneous tumor and that genetic profiling is mandatory for the development of effective personalized therapeutic approaches for melanoma patients.

No MeSH data available.


Related in: MedlinePlus

ERβ agonists significantly and specifically inhibit the proliferation of BLM melanoma cells.(A) BLM cells were treated with different doses of the classical ERβ agonist DPN every 48 h for three times. DPN significantly decreased cell proliferation at the dose of 10−8 M. (B) Similar results were obtained when the cells were treated with E2. (C) The antiproliferative effect of both ERβ ligands (10−8 M) was found to be specific since it was completely abrogated by cotreatment of the cells with the ER antagonist ICI 182,780 (10−6 M). (D) BLM cells were treated with KB1, KB2, or KB4 (10−9, 10−8, 10−7 M) every 48 h for three times. All three ERβ ligands significantly reduced cell proliferation at the dose of 10−8 M. (E) BLM cells were treated with KB1, at different doses (109−10−7 M). The ERβ agonists decreased cell growth, being significantly effective at the dose of 10−8 M. (F) The antiproliferative activity of KB1 (10−8 M) was found to be specific since it was completely abrogated by cotreatment of the cells with the ER antagonist ICI 182,780. Each experimental group consisted of six replicates and each experiment was repeated three-five times. Results are given as cell number/plate. Data represent mean values ± SEM. *P<0.05. C, controls.
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pone.0134396.g002: ERβ agonists significantly and specifically inhibit the proliferation of BLM melanoma cells.(A) BLM cells were treated with different doses of the classical ERβ agonist DPN every 48 h for three times. DPN significantly decreased cell proliferation at the dose of 10−8 M. (B) Similar results were obtained when the cells were treated with E2. (C) The antiproliferative effect of both ERβ ligands (10−8 M) was found to be specific since it was completely abrogated by cotreatment of the cells with the ER antagonist ICI 182,780 (10−6 M). (D) BLM cells were treated with KB1, KB2, or KB4 (10−9, 10−8, 10−7 M) every 48 h for three times. All three ERβ ligands significantly reduced cell proliferation at the dose of 10−8 M. (E) BLM cells were treated with KB1, at different doses (109−10−7 M). The ERβ agonists decreased cell growth, being significantly effective at the dose of 10−8 M. (F) The antiproliferative activity of KB1 (10−8 M) was found to be specific since it was completely abrogated by cotreatment of the cells with the ER antagonist ICI 182,780. Each experimental group consisted of six replicates and each experiment was repeated three-five times. Results are given as cell number/plate. Data represent mean values ± SEM. *P<0.05. C, controls.

Mentions: Experiments were first performed to investigate the effects of ERβ activation on the growth of BLM melanoma cells, expressing ERβ. The selective ERβ agonist DPN decreased BLM cell proliferation at the concentrations of 10−9 and 5x10-9 M, being significantly effective at the dose of 10−8 M (Fig 2A). This significant effect was followed by a decline at concentrations of 5x10-8 and 10−7 M. Accordingly, the natural estrogenic ligand E2 exerted a significant antiproliferative effect on BLM cell proliferation at the concentration of 10−8 M (Fig 2B). The antiproliferative activity of both DPN and E2 (10−8 M) was completely counteracted by cotreatment of the cells with the ER antagonist ICI 182,780 (10−6 M) (Fig 2C).


Estrogen Receptor β Agonists Differentially Affect the Growth of Human Melanoma Cell Lines.

Marzagalli M, Casati L, Moretti RM, Montagnani Marelli M, Limonta P - PLoS ONE (2015)

ERβ agonists significantly and specifically inhibit the proliferation of BLM melanoma cells.(A) BLM cells were treated with different doses of the classical ERβ agonist DPN every 48 h for three times. DPN significantly decreased cell proliferation at the dose of 10−8 M. (B) Similar results were obtained when the cells were treated with E2. (C) The antiproliferative effect of both ERβ ligands (10−8 M) was found to be specific since it was completely abrogated by cotreatment of the cells with the ER antagonist ICI 182,780 (10−6 M). (D) BLM cells were treated with KB1, KB2, or KB4 (10−9, 10−8, 10−7 M) every 48 h for three times. All three ERβ ligands significantly reduced cell proliferation at the dose of 10−8 M. (E) BLM cells were treated with KB1, at different doses (109−10−7 M). The ERβ agonists decreased cell growth, being significantly effective at the dose of 10−8 M. (F) The antiproliferative activity of KB1 (10−8 M) was found to be specific since it was completely abrogated by cotreatment of the cells with the ER antagonist ICI 182,780. Each experimental group consisted of six replicates and each experiment was repeated three-five times. Results are given as cell number/plate. Data represent mean values ± SEM. *P<0.05. C, controls.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4520550&req=5

pone.0134396.g002: ERβ agonists significantly and specifically inhibit the proliferation of BLM melanoma cells.(A) BLM cells were treated with different doses of the classical ERβ agonist DPN every 48 h for three times. DPN significantly decreased cell proliferation at the dose of 10−8 M. (B) Similar results were obtained when the cells were treated with E2. (C) The antiproliferative effect of both ERβ ligands (10−8 M) was found to be specific since it was completely abrogated by cotreatment of the cells with the ER antagonist ICI 182,780 (10−6 M). (D) BLM cells were treated with KB1, KB2, or KB4 (10−9, 10−8, 10−7 M) every 48 h for three times. All three ERβ ligands significantly reduced cell proliferation at the dose of 10−8 M. (E) BLM cells were treated with KB1, at different doses (109−10−7 M). The ERβ agonists decreased cell growth, being significantly effective at the dose of 10−8 M. (F) The antiproliferative activity of KB1 (10−8 M) was found to be specific since it was completely abrogated by cotreatment of the cells with the ER antagonist ICI 182,780. Each experimental group consisted of six replicates and each experiment was repeated three-five times. Results are given as cell number/plate. Data represent mean values ± SEM. *P<0.05. C, controls.
Mentions: Experiments were first performed to investigate the effects of ERβ activation on the growth of BLM melanoma cells, expressing ERβ. The selective ERβ agonist DPN decreased BLM cell proliferation at the concentrations of 10−9 and 5x10-9 M, being significantly effective at the dose of 10−8 M (Fig 2A). This significant effect was followed by a decline at concentrations of 5x10-8 and 10−7 M. Accordingly, the natural estrogenic ligand E2 exerted a significant antiproliferative effect on BLM cell proliferation at the concentration of 10−8 M (Fig 2B). The antiproliferative activity of both DPN and E2 (10−8 M) was completely counteracted by cotreatment of the cells with the ER antagonist ICI 182,780 (10−6 M) (Fig 2C).

Bottom Line: Moreover, the antiproliferative activity of ERβ agonists was associated with an altered expression of G1-S transition-related proteins.In these cells, global DNA was found to be hypomethylated when compared to normal melanocytes; this DNA hypomethylation status was reverted by ERβ activation.ERβ agonists also decreased the proliferation of WM115 (BRAF V600D-mutant) cells, while they failed to reduce the growth of A375 and WM1552 (BRAF V600E-mutant) cells.

View Article: PubMed Central - PubMed

Affiliation: Department of Pharmacological and Biomolecular Sciences, Università degli Studi di Milano, Milano, Italy.

ABSTRACT

Background: Cutaneous melanoma is an aggressive malignancy; its incidence is increasing worldwide and its prognosis remains poor. Clinical observations indicate that estrogen receptor β (ERβ) is expressed in melanoma tissues and its expression decreases with tumor progression, suggesting its tumor suppressive function. These experiments were performed to investigate the effects of ERβ activation on melanoma cell growth.

Methods and results: Protein expression was analyzed by Western blot and immunofluorescence assays. Cell proliferation was assessed by counting the cells by hemocytometer. ERβ transcriptional activity was evaluated by gene reporter assay. Global DNA methylation was analyzed by restriction enzyme assay and ERβ isoforms were identified by qRT-PCR. We demonstrated that ERβ is expressed in a panel of human melanoma cell lines (BLM, WM115, A375, WM1552). In BLM (NRAS-mutant) cells, ERβ agonists significantly and specifically inhibited cell proliferation. ERβ activation triggered its cytoplasmic-to-nuclear translocation and transcriptional activity. Moreover, the antiproliferative activity of ERβ agonists was associated with an altered expression of G1-S transition-related proteins. In these cells, global DNA was found to be hypomethylated when compared to normal melanocytes; this DNA hypomethylation status was reverted by ERβ activation. ERβ agonists also decreased the proliferation of WM115 (BRAF V600D-mutant) cells, while they failed to reduce the growth of A375 and WM1552 (BRAF V600E-mutant) cells. Finally, we could observe that ERβ isoforms are expressed at different levels in the various cell lines. Specific oncogenic mutations or differential expression of receptor isoforms might be responsible for the different responses of cell lines to ERβ agonists.

Conclusions: Our results demonstrate that ERβ is expressed in melanoma cell lines and that ERβ agonists differentially regulate the proliferation of these cells. These data confirm the notion that melanoma is a heterogeneous tumor and that genetic profiling is mandatory for the development of effective personalized therapeutic approaches for melanoma patients.

No MeSH data available.


Related in: MedlinePlus