Limits...
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 differentially affect the proliferation of melanoma cell lines.(A) IGR-39, A375 and WM1552 melanoma cells were treated with DPN (10−9–10−7 M) every 48 h for three times. No effect on cell proliferation could be observed in any cell line tested. (B) WM115 cells were treated with DPN, E2, or KB1 (10−10–10−7 M) every 48 h for three times. DPN was significantly effective in decreasing cell proliferation at the doses of 10−9 and 10−8 M. On the other hand, both E2 and KB1 significantly reduced cell proliferation at the dose of 10−8 M. Each experimental group consisted of six replicates and each experiment was repeated three times. Results are given as cell number/plate. Data represent mean values ± SEM. *P<0.05. C, controls.
© Copyright Policy
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4520550&req=5

pone.0134396.g006: ERβ agonists differentially affect the proliferation of melanoma cell lines.(A) IGR-39, A375 and WM1552 melanoma cells were treated with DPN (10−9–10−7 M) every 48 h for three times. No effect on cell proliferation could be observed in any cell line tested. (B) WM115 cells were treated with DPN, E2, or KB1 (10−10–10−7 M) every 48 h for three times. DPN was significantly effective in decreasing cell proliferation at the doses of 10−9 and 10−8 M. On the other hand, both E2 and KB1 significantly reduced cell proliferation at the dose of 10−8 M. Each experimental group consisted of six replicates and each experiment was repeated three times. Results are given as cell number/plate. Data represent mean values ± SEM. *P<0.05. C, controls.

Mentions: Based on the results obtained in BLM melanoma cells (expressing the ERβ receptor subtype and harboring the NRAS mutation), further experiments were performed to assess the effects of ERβ activation on the proliferation of different melanoma cell lines, either lacking the expression of ERβ or expressing ERβ while harboring different oncogenic mutations (e.g., BRAF). Specifically, the effects of ERβ agonists were assessed on the proliferation of the following human melanoma cell lines: IGR-39 cells (expressing almost undetectable levels of ERβ), A375 and WM1552 cells (expressing ERβ and harboring the BRAF V600E mutation), and WM115 cells (expressing ERβ and harboring the rare BRAF V600D mutation). IGR-39, A375 and WM1552 cells were treated with DPN (10−9–10−7 M) while WM115 cells were treated with DPN, E2 and KB1 (10−10–10−7 M), as described for BLM cells. As expected, we found that DPN does not affect the proliferation of IGR-39 cells, lacking ERβ expression (Fig 6A). Unexpectedly, and interestingly, the ERβ agonist also failed to affect the growth of A375 and WM1552 melanoma cells, expressing the receptor subtype (Fig 6B and 6C). On the other hand, the proliferation of WM115 cells was reduced by the treatment with DPN, E2 and KB1, with a dose-response curve similar to that observed in BLM cells (Fig 6D). Taken together, these results indicate that ERβ activation differentially affects the proliferation of melanoma cell lines. The reasons for these observations are still unclear; however, we might speculate that the efficacy of ERβ agonists in reducing melanoma growth might depend not only on the presence of the receptor but also on other particular features of each melanoma, such as the oncogenic mutation status (NRAS, BRAF) of the tumor.


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 differentially affect the proliferation of melanoma cell lines.(A) IGR-39, A375 and WM1552 melanoma cells were treated with DPN (10−9–10−7 M) every 48 h for three times. No effect on cell proliferation could be observed in any cell line tested. (B) WM115 cells were treated with DPN, E2, or KB1 (10−10–10−7 M) every 48 h for three times. DPN was significantly effective in decreasing cell proliferation at the doses of 10−9 and 10−8 M. On the other hand, both E2 and KB1 significantly reduced cell proliferation at the dose of 10−8 M. Each experimental group consisted of six replicates and each experiment was repeated three times. Results are given as cell number/plate. Data represent mean values ± SEM. *P<0.05. C, controls.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4520550&req=5

pone.0134396.g006: ERβ agonists differentially affect the proliferation of melanoma cell lines.(A) IGR-39, A375 and WM1552 melanoma cells were treated with DPN (10−9–10−7 M) every 48 h for three times. No effect on cell proliferation could be observed in any cell line tested. (B) WM115 cells were treated with DPN, E2, or KB1 (10−10–10−7 M) every 48 h for three times. DPN was significantly effective in decreasing cell proliferation at the doses of 10−9 and 10−8 M. On the other hand, both E2 and KB1 significantly reduced cell proliferation at the dose of 10−8 M. Each experimental group consisted of six replicates and each experiment was repeated three times. Results are given as cell number/plate. Data represent mean values ± SEM. *P<0.05. C, controls.
Mentions: Based on the results obtained in BLM melanoma cells (expressing the ERβ receptor subtype and harboring the NRAS mutation), further experiments were performed to assess the effects of ERβ activation on the proliferation of different melanoma cell lines, either lacking the expression of ERβ or expressing ERβ while harboring different oncogenic mutations (e.g., BRAF). Specifically, the effects of ERβ agonists were assessed on the proliferation of the following human melanoma cell lines: IGR-39 cells (expressing almost undetectable levels of ERβ), A375 and WM1552 cells (expressing ERβ and harboring the BRAF V600E mutation), and WM115 cells (expressing ERβ and harboring the rare BRAF V600D mutation). IGR-39, A375 and WM1552 cells were treated with DPN (10−9–10−7 M) while WM115 cells were treated with DPN, E2 and KB1 (10−10–10−7 M), as described for BLM cells. As expected, we found that DPN does not affect the proliferation of IGR-39 cells, lacking ERβ expression (Fig 6A). Unexpectedly, and interestingly, the ERβ agonist also failed to affect the growth of A375 and WM1552 melanoma cells, expressing the receptor subtype (Fig 6B and 6C). On the other hand, the proliferation of WM115 cells was reduced by the treatment with DPN, E2 and KB1, with a dose-response curve similar to that observed in BLM cells (Fig 6D). Taken together, these results indicate that ERβ activation differentially affects the proliferation of melanoma cell lines. The reasons for these observations are still unclear; however, we might speculate that the efficacy of ERβ agonists in reducing melanoma growth might depend not only on the presence of the receptor but also on other particular features of each melanoma, such as the oncogenic mutation status (NRAS, BRAF) of the tumor.

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