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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 ligands trigger cytoplasmic-to-nuclear translocation of ERβ and induce its transcriptional acitivity in BLM melanoma cells.(A) Immunofluorescence assay of ERβ intracellular localization. In control BLM melanoma cells, ERβ is mainly localized at the cytoplasmic level. Treatment of the cells with either DPN or E2 (10−8 M, for 24 h) induces ERβ translocation into the nucleus. A representative picture from three experiments done independently, which gave the same results, is reported. (B) The transcriptional activity of the ERβ protein in BLM cells was analyzed using the pVERE-tk-LUC plasmid (cotransfected with pCMVβ). The results were normalized for β-galactosidase activity. Treatment of the cells with either DPN or E2 (10−8 M, for 24 h) significantly increased ERβ transcriptional activity. Each experimental group consisted of three replicates and each experiment was repeated three times. Data represent mean values ± SEM. *P<0.05. C, controls.
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pone.0134396.g003: ERβ ligands trigger cytoplasmic-to-nuclear translocation of ERβ and induce its transcriptional acitivity in BLM melanoma cells.(A) Immunofluorescence assay of ERβ intracellular localization. In control BLM melanoma cells, ERβ is mainly localized at the cytoplasmic level. Treatment of the cells with either DPN or E2 (10−8 M, for 24 h) induces ERβ translocation into the nucleus. A representative picture from three experiments done independently, which gave the same results, is reported. (B) The transcriptional activity of the ERβ protein in BLM cells was analyzed using the pVERE-tk-LUC plasmid (cotransfected with pCMVβ). The results were normalized for β-galactosidase activity. Treatment of the cells with either DPN or E2 (10−8 M, for 24 h) significantly increased ERβ transcriptional activity. Each experimental group consisted of three replicates and each experiment was repeated three times. Data represent mean values ± SEM. *P<0.05. C, controls.

Mentions: Experiments were performed to verify whether, in BLM melanoma cells, ERβ might function according to the classical model of estrogen action [11]. By immunofluorescence analysis, we could show that, in BLM cells, most of the ERβ staining was confined in the cytoplasm (Fig 3A); treatment of melanoma cells (24 h) with both DPN and E2 (10−8 M) induced its nuclear translocation (Fig 3A). Then, we analyzed the effects of ERβ ligands on the transcriptional activity of the receptor in melanoma cells. Fig 3B shows that treatment of BLM cells with either DPN or E2 for 24 h significantly increased the transcriptional activation of the ERE-Luc reporter plasmid (normalized for β-galactosidase) indicating that, in BLM melanoma cells, ERβ is associated with the classical transcriptional activity at the nuclear level.


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 ligands trigger cytoplasmic-to-nuclear translocation of ERβ and induce its transcriptional acitivity in BLM melanoma cells.(A) Immunofluorescence assay of ERβ intracellular localization. In control BLM melanoma cells, ERβ is mainly localized at the cytoplasmic level. Treatment of the cells with either DPN or E2 (10−8 M, for 24 h) induces ERβ translocation into the nucleus. A representative picture from three experiments done independently, which gave the same results, is reported. (B) The transcriptional activity of the ERβ protein in BLM cells was analyzed using the pVERE-tk-LUC plasmid (cotransfected with pCMVβ). The results were normalized for β-galactosidase activity. Treatment of the cells with either DPN or E2 (10−8 M, for 24 h) significantly increased ERβ transcriptional activity. Each experimental group consisted of three replicates and each experiment was repeated three times. Data represent mean values ± SEM. *P<0.05. C, controls.
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Related In: Results  -  Collection

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pone.0134396.g003: ERβ ligands trigger cytoplasmic-to-nuclear translocation of ERβ and induce its transcriptional acitivity in BLM melanoma cells.(A) Immunofluorescence assay of ERβ intracellular localization. In control BLM melanoma cells, ERβ is mainly localized at the cytoplasmic level. Treatment of the cells with either DPN or E2 (10−8 M, for 24 h) induces ERβ translocation into the nucleus. A representative picture from three experiments done independently, which gave the same results, is reported. (B) The transcriptional activity of the ERβ protein in BLM cells was analyzed using the pVERE-tk-LUC plasmid (cotransfected with pCMVβ). The results were normalized for β-galactosidase activity. Treatment of the cells with either DPN or E2 (10−8 M, for 24 h) significantly increased ERβ transcriptional activity. Each experimental group consisted of three replicates and each experiment was repeated three times. Data represent mean values ± SEM. *P<0.05. C, controls.
Mentions: Experiments were performed to verify whether, in BLM melanoma cells, ERβ might function according to the classical model of estrogen action [11]. By immunofluorescence analysis, we could show that, in BLM cells, most of the ERβ staining was confined in the cytoplasm (Fig 3A); treatment of melanoma cells (24 h) with both DPN and E2 (10−8 M) induced its nuclear translocation (Fig 3A). Then, we analyzed the effects of ERβ ligands on the transcriptional activity of the receptor in melanoma cells. Fig 3B shows that treatment of BLM cells with either DPN or E2 for 24 h significantly increased the transcriptional activation of the ERE-Luc reporter plasmid (normalized for β-galactosidase) indicating that, in BLM melanoma cells, ERβ is associated with the classical transcriptional activity at the nuclear level.

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