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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, but not ERα, is expressed in human melanoma cells.(A) As a positive control, the expression of ERβ was evaluated by Western blot analysis in human BLM melanoma cells engineered to overexpress the receptor subtype protein, utilizing two primary antibodies: H-150 (Santa Cruz) and 14C8 (Abcam). A band corresponding to the receptor protein (59 kDa) was detected in basal conditions, both in control (C) and in Lipofectamine (Lipo) treated BLM cells. As expected, the intensity of this band was found to be significantly increased after ERβ overexpression (24–72 h), with the highest level of expression at 24 h. (B) By Western blot analysis, utilizing the two primary antibodies H-150 and 14C8, ERβ was found to be expressed at high levels in human BLM, A375, WM115, WM1552 melanoma cell lines (lanes 3, 4, 5, 7), while the human IGR-39 melanoma cell line expressed the receptor at almost undetectable levels (lane 6). ERβ was also expressed in human MCF-7 breast cancer cells, utilized as a positive control (lane 1), but it was not expressed in the human HEK293, utilized as a negative control. (C) On the other hand, all the human melanoma cells lines tested (lanes 2–6) did not express the ERα receptor isoform, which was expressed only in the control cell line (MCF-7, lane 1). β-actin was utilized as a loading control. For each analysis, one representative of three different experiments, which gave similar results, is shown.
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pone.0134396.g001: ERβ, but not ERα, is expressed in human melanoma cells.(A) As a positive control, the expression of ERβ was evaluated by Western blot analysis in human BLM melanoma cells engineered to overexpress the receptor subtype protein, utilizing two primary antibodies: H-150 (Santa Cruz) and 14C8 (Abcam). A band corresponding to the receptor protein (59 kDa) was detected in basal conditions, both in control (C) and in Lipofectamine (Lipo) treated BLM cells. As expected, the intensity of this band was found to be significantly increased after ERβ overexpression (24–72 h), with the highest level of expression at 24 h. (B) By Western blot analysis, utilizing the two primary antibodies H-150 and 14C8, ERβ was found to be expressed at high levels in human BLM, A375, WM115, WM1552 melanoma cell lines (lanes 3, 4, 5, 7), while the human IGR-39 melanoma cell line expressed the receptor at almost undetectable levels (lane 6). ERβ was also expressed in human MCF-7 breast cancer cells, utilized as a positive control (lane 1), but it was not expressed in the human HEK293, utilized as a negative control. (C) On the other hand, all the human melanoma cells lines tested (lanes 2–6) did not express the ERα receptor isoform, which was expressed only in the control cell line (MCF-7, lane 1). β-actin was utilized as a loading control. For each analysis, one representative of three different experiments, which gave similar results, is shown.

Mentions: The expression of ERβ in human melanoma cell lines was analyzed by Western blot assay utilizing two primary antibodies: H-150 (Santa Cruz Biotechnology) and 14C8 (Abcam). First, in order to obtain an appropriate positive control, ERβ was evaluated in BLM melanoma cells engineered to overexpress the receptor protein. Fig 1A shows that a protein band, corresponding to the molecular weight of 59 kDa, is expressed in BLM cells either in normal culture conditions (C) or in the presence of Lipofectamine (Lipo), at 24–72 h. More importantly, Fig 1A also shows that the expression levels of this protein band are sharply increased in BLM cells overexpressing the receptor (ERβ) at 24 after transfection, and slightly decrease at 48 and 72 h. These data, obtained with the two different antibodies, confirm that the molecular weight of this receptor subtype corresponds to 59 kDa, as previously reported [43,44].


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, but not ERα, is expressed in human melanoma cells.(A) As a positive control, the expression of ERβ was evaluated by Western blot analysis in human BLM melanoma cells engineered to overexpress the receptor subtype protein, utilizing two primary antibodies: H-150 (Santa Cruz) and 14C8 (Abcam). A band corresponding to the receptor protein (59 kDa) was detected in basal conditions, both in control (C) and in Lipofectamine (Lipo) treated BLM cells. As expected, the intensity of this band was found to be significantly increased after ERβ overexpression (24–72 h), with the highest level of expression at 24 h. (B) By Western blot analysis, utilizing the two primary antibodies H-150 and 14C8, ERβ was found to be expressed at high levels in human BLM, A375, WM115, WM1552 melanoma cell lines (lanes 3, 4, 5, 7), while the human IGR-39 melanoma cell line expressed the receptor at almost undetectable levels (lane 6). ERβ was also expressed in human MCF-7 breast cancer cells, utilized as a positive control (lane 1), but it was not expressed in the human HEK293, utilized as a negative control. (C) On the other hand, all the human melanoma cells lines tested (lanes 2–6) did not express the ERα receptor isoform, which was expressed only in the control cell line (MCF-7, lane 1). β-actin was utilized as a loading control. For each analysis, one representative of three different experiments, which gave similar results, is shown.
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pone.0134396.g001: ERβ, but not ERα, is expressed in human melanoma cells.(A) As a positive control, the expression of ERβ was evaluated by Western blot analysis in human BLM melanoma cells engineered to overexpress the receptor subtype protein, utilizing two primary antibodies: H-150 (Santa Cruz) and 14C8 (Abcam). A band corresponding to the receptor protein (59 kDa) was detected in basal conditions, both in control (C) and in Lipofectamine (Lipo) treated BLM cells. As expected, the intensity of this band was found to be significantly increased after ERβ overexpression (24–72 h), with the highest level of expression at 24 h. (B) By Western blot analysis, utilizing the two primary antibodies H-150 and 14C8, ERβ was found to be expressed at high levels in human BLM, A375, WM115, WM1552 melanoma cell lines (lanes 3, 4, 5, 7), while the human IGR-39 melanoma cell line expressed the receptor at almost undetectable levels (lane 6). ERβ was also expressed in human MCF-7 breast cancer cells, utilized as a positive control (lane 1), but it was not expressed in the human HEK293, utilized as a negative control. (C) On the other hand, all the human melanoma cells lines tested (lanes 2–6) did not express the ERα receptor isoform, which was expressed only in the control cell line (MCF-7, lane 1). β-actin was utilized as a loading control. For each analysis, one representative of three different experiments, which gave similar results, is shown.
Mentions: The expression of ERβ in human melanoma cell lines was analyzed by Western blot assay utilizing two primary antibodies: H-150 (Santa Cruz Biotechnology) and 14C8 (Abcam). First, in order to obtain an appropriate positive control, ERβ was evaluated in BLM melanoma cells engineered to overexpress the receptor protein. Fig 1A shows that a protein band, corresponding to the molecular weight of 59 kDa, is expressed in BLM cells either in normal culture conditions (C) or in the presence of Lipofectamine (Lipo), at 24–72 h. More importantly, Fig 1A also shows that the expression levels of this protein band are sharply increased in BLM cells overexpressing the receptor (ERβ) at 24 after transfection, and slightly decrease at 48 and 72 h. These data, obtained with the two different antibodies, confirm that the molecular weight of this receptor subtype corresponds to 59 kDa, as previously reported [43,44].

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