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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 activation induces global DNA methylation reprogramming in BLM melanoma cells.(A) Preliminary experiments were carried out to analyze the global DNA methylation status of BLM cells when compared to that of human normal melanocytes (hMel). To this purpose, a restriction enzymatic assay was employed. For each DNA sample, two restriction digests were performed: one with RsaI and MspI, and one with RsaI and HpaII. RsaI is methylation insensitive, while MspI and HpaII are sensitive to DNA methylation and are able to cut only unmethylated restriction sites. The digests were then amplified by PCR. Data are expressed as the MspI/RsaI or HpaII/RsaI ratios relative to the intensity of the bands. BLM melanoma cells were found to be globally hypomethylated when compared to normal melanocytes, when both MspI and HpaII restriction enzymes were utilized. One representative of three different experiments, which gave similar results, is reported. (B) Experiments were performed to evaluate whether activation of ERβ might affect the global DNA hypomethylation status observed in melanoma cells. BLM cells were treated with either DPN or E2 (10−8M) for 24 or 48 h; the DNA methylation status was then evaluated as described above. Both DPN (at 24 and 48 h) and E2 (at 24 h) increased the DNA methylation profile of BLM cells, indicating that ERβ activation reverts the DNA hypomethylation status in melanoma cells. One representative of three different experiments, which gave similar results, is reported.
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pone.0134396.g005: ERβ activation induces global DNA methylation reprogramming in BLM melanoma cells.(A) Preliminary experiments were carried out to analyze the global DNA methylation status of BLM cells when compared to that of human normal melanocytes (hMel). To this purpose, a restriction enzymatic assay was employed. For each DNA sample, two restriction digests were performed: one with RsaI and MspI, and one with RsaI and HpaII. RsaI is methylation insensitive, while MspI and HpaII are sensitive to DNA methylation and are able to cut only unmethylated restriction sites. The digests were then amplified by PCR. Data are expressed as the MspI/RsaI or HpaII/RsaI ratios relative to the intensity of the bands. BLM melanoma cells were found to be globally hypomethylated when compared to normal melanocytes, when both MspI and HpaII restriction enzymes were utilized. One representative of three different experiments, which gave similar results, is reported. (B) Experiments were performed to evaluate whether activation of ERβ might affect the global DNA hypomethylation status observed in melanoma cells. BLM cells were treated with either DPN or E2 (10−8M) for 24 or 48 h; the DNA methylation status was then evaluated as described above. Both DPN (at 24 and 48 h) and E2 (at 24 h) increased the DNA methylation profile of BLM cells, indicating that ERβ activation reverts the DNA hypomethylation status in melanoma cells. One representative of three different experiments, which gave similar results, is reported.

Mentions: Experiments were first carried out to analyze the global DNA methylation status of human BLM melanoma cells when compared to that of human melanocytes. To this purpose, a restriction enzymatic assay, utilizing the two methylation sensitive restriction enzymes MspI and HpaII, was performed. These enzymes recognize the same tetranucleotide sequence (5'-CCGG-3') but display different sensitivity to DNA methylation. In particular, MspI does not cut when the external cytosine is methylated while HpaII does not cut when any of the two cytosines is methylated [39,40]. Fig 5A shows that BLM cells are globally hypomethylated when compared to human melanocytes (hMEL), when both MspI and HpaII restriction enzymes are utilized. These data confirm that melanoma cells are characterized by an aberrant global DNA hypomethylation, which is known to be associated with genome instability.


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 activation induces global DNA methylation reprogramming in BLM melanoma cells.(A) Preliminary experiments were carried out to analyze the global DNA methylation status of BLM cells when compared to that of human normal melanocytes (hMel). To this purpose, a restriction enzymatic assay was employed. For each DNA sample, two restriction digests were performed: one with RsaI and MspI, and one with RsaI and HpaII. RsaI is methylation insensitive, while MspI and HpaII are sensitive to DNA methylation and are able to cut only unmethylated restriction sites. The digests were then amplified by PCR. Data are expressed as the MspI/RsaI or HpaII/RsaI ratios relative to the intensity of the bands. BLM melanoma cells were found to be globally hypomethylated when compared to normal melanocytes, when both MspI and HpaII restriction enzymes were utilized. One representative of three different experiments, which gave similar results, is reported. (B) Experiments were performed to evaluate whether activation of ERβ might affect the global DNA hypomethylation status observed in melanoma cells. BLM cells were treated with either DPN or E2 (10−8M) for 24 or 48 h; the DNA methylation status was then evaluated as described above. Both DPN (at 24 and 48 h) and E2 (at 24 h) increased the DNA methylation profile of BLM cells, indicating that ERβ activation reverts the DNA hypomethylation status in melanoma cells. One representative of three different experiments, which gave similar results, is reported.
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pone.0134396.g005: ERβ activation induces global DNA methylation reprogramming in BLM melanoma cells.(A) Preliminary experiments were carried out to analyze the global DNA methylation status of BLM cells when compared to that of human normal melanocytes (hMel). To this purpose, a restriction enzymatic assay was employed. For each DNA sample, two restriction digests were performed: one with RsaI and MspI, and one with RsaI and HpaII. RsaI is methylation insensitive, while MspI and HpaII are sensitive to DNA methylation and are able to cut only unmethylated restriction sites. The digests were then amplified by PCR. Data are expressed as the MspI/RsaI or HpaII/RsaI ratios relative to the intensity of the bands. BLM melanoma cells were found to be globally hypomethylated when compared to normal melanocytes, when both MspI and HpaII restriction enzymes were utilized. One representative of three different experiments, which gave similar results, is reported. (B) Experiments were performed to evaluate whether activation of ERβ might affect the global DNA hypomethylation status observed in melanoma cells. BLM cells were treated with either DPN or E2 (10−8M) for 24 or 48 h; the DNA methylation status was then evaluated as described above. Both DPN (at 24 and 48 h) and E2 (at 24 h) increased the DNA methylation profile of BLM cells, indicating that ERβ activation reverts the DNA hypomethylation status in melanoma cells. One representative of three different experiments, which gave similar results, is reported.
Mentions: Experiments were first carried out to analyze the global DNA methylation status of human BLM melanoma cells when compared to that of human melanocytes. To this purpose, a restriction enzymatic assay, utilizing the two methylation sensitive restriction enzymes MspI and HpaII, was performed. These enzymes recognize the same tetranucleotide sequence (5'-CCGG-3') but display different sensitivity to DNA methylation. In particular, MspI does not cut when the external cytosine is methylated while HpaII does not cut when any of the two cytosines is methylated [39,40]. Fig 5A shows that BLM cells are globally hypomethylated when compared to human melanocytes (hMEL), when both MspI and HpaII restriction enzymes are utilized. These data confirm that melanoma cells are characterized by an aberrant global DNA hypomethylation, which is known to be associated with genome instability.

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