Different Susceptibilities of Human Melanoma Cell Lines to G2/M Blockage and Cell Death Activation in Response to the Estrogen Receptor β agonist LY500307

Abstract

Background: Gender differences in melanoma incidence, metastasis formation and disease progression are increasingly evident in epidemiological studies, with women showing significantly better survival than men. Among factors possibly underlying the disparities, sex hormones seem to play a key role. Nonetheless, functional mechanisms are still unclear, except for the antitumor ability of Estrogen Receptor (ER) β, whose expression determination has often been suggested for melanoma prognosis. In this study, we aimed at evaluating the molecular mechanisms and functional effects associated with ERβ signaling by using its agonist LY500307. Methods: We evaluated the antitumor effect of the specific synthetic ERβ agonist LY500307 on some human melanoma cell lines, selected for different genetic background, expression levels of ERs and tumor progression. The expression of α and β estrogen receptors was investigated taking advantage of The Cancer Genome Atlas database and confirmed on some selected melanoma cell lines. The biological effects of LY500307 were determined in vitro looking at melanoma cell proliferation, cell cycle profiles and migration demonstrating by western blot the involvement of some pathway specific markers. The LY500307-dependent induction of cell death was also analyzed by flow cytometry and western blot analysis of caspase 3 and poly adenosine diphosphate-ribose polymerase (PARP). Results: A significant decrease in the expression of both ERs, even more pronounced for ERα, has been found in patients with metastatic NRAS mutation. Treatment with LY500307 significantly reduced the proliferation of melanoma cells showing a cell cycle arrest at the G2/M boundary phase and promoting apoptosis with different sensitivities associated with disease stage and mutation. Indeed, the ERβ agonist affects melanoma migration, inducing a reversion of the epithelial-mesenchymal transition, more evident in a low aggressive primary melanoma cell line. Conclusion: These results demonstrate the capability of LY500307 to reduce melanoma malignancy, counteracting cell viability and dissemination, overall suggesting a possible future use of LY500307 in personalized combined therapy.

Keywords: Estrogen Receptors; LY500307; apoptosis.; cell cycle; melanoma.

Figure 1

ERα and ERβ expression in SKCM patients. RNA-seq counts in logarithmic scale in total SKCM, BRAF and NRAS mutated patients with primary or metastatic tumors for ERα gene expression (ESR1), ERβ gene expression (ESR2) and ratio between ERα and ERβ gene expressions. Data are reported as interquartile (IQ) boxplots with inbox lines indicating median values and whiskers ± 1.5 IQ range. Asterisks indicate the level of significance: **p < 0.01; ***p<0.001.

Figure 2

ERα and ERβ expression levels in different tumor staged melanoma cell lines. Western blot analysis of (A) ERα and (B) ERβ in primary (Me1007, Mel501, WM983A), recurrence (Me1402/R) and metastatic (A375M, Me665/1, SK-MEL-30) melanoma cell lines, and corresponding relative densitometric quantification. β-Actin was utilized as internal loading control. Data are expressed as the mean + SD of three independent analyses.

Figure 3

LY500307 and its combined treatment with ERβ/α antagonists (PHTPP, MPP) on melanoma cell cycle. Cell cycle analysis of control (CTR) vs cells treated with increased concentrations of LY500307 (2μM, 4μM, 8μM) in (A) Me1402/R, (B) Me665/1 and (C) A375M cell lines for 24 hours. Cell cycle analysis of Me665/1 cells treated with either (D) PHTPP (5μM) and LY500307 (4μM or 8μM) or (E) MPP (5μM) and LY500307 (2μM) alone or in combination for 24 hours respect to the untreated control (CTR). Data are represented as mean ± SD of three independent experiments. Asterisks indicate the level of significance: * p<0.05; ** p<0.01; *** p<0.001 compared with the corresponding CTR or LY500307 treated cells.

Figure 4

Evaluation of cell cycle regulators. Representative Western Blots of p21 and cyclin B1 expression levels in Me1402/R (A), Me665/1 (B) and A375M (C) cells treated with increased concentrations of LY500307 (2μM, 4μM, 8μM) for 24 hours. β-Actin and Tubulin were utilized as internal loading control. Densitometric quantifications shown as fold increase are represented as mean + SD of three independent experiments. Asterisks indicate the level of significance: * p<0.05; ** p<0.01; *** p<0.001 compared with the control cells (CTR).

Figure 5

LY500307 treatment induces alteration in mitotic figures. Confocal microscopy visualization of cellular and nuclear morphology of (A) Me1402/R, (B) Me665/1 and (C) A375M melanoma cell lines after 12, 5 and 24 hours of LY500307 treatment, respectively. Cells were stained with Phalloidin (Alexa Fluor 647-red) and α-tubulin (Alexa Fluor488-green) for visualization of actin and tubulin filaments, respectively. Nuclei were counter-stained with Hoechst 33342 (blue). Scale bar: 10μM

Figure 6

LY500307 treatment modulates the expression levels of DNA damage related proteins. Representative Western Blot analysis of Wee1 and p-H2AX expression levels after 24 hours of treatment with increased concentrations of LY500307 (2μM, 4μM, 8μM) in (A) Me1402/R, (B) Me665/1 and (C) A375M cells. β-Actin was utilized as internal loading control. Densitometric quantifications, shown as fold increase, are represented as mean + SD of three independent experiments. Asterisks indicate the level of significance: * p<0.05, ** p<0.01, *** p<0.001 compared with the control cells (CTR).

Figure 7

LY500307 induces apoptosis in Me1402R cell line. Representative Western Blots illustrate expression levels of Caspase 3, PARP and their cleaved forms in Me1402/R (A), Me665/1 (C) and A375M (D) melanoma cells exposed to increased concentrations of LY500307 (2μM, 4μM, 8μM) for 24 hours. Densitometric quantifications of Cleaved/Total Caspase-3 and PARP ratios are represented as mean + SD of three independent experiments. Quantification of apoptotic cell fractions of Me1402R and Me665/1 (B) cells after 24 hours of treatment with either vehicle or LY500307 (2μM, 4μM, 8μM), by Annexin V-FITC and Propidium Iodide (PI) staining and FACS analysis. A representative experiment out of three is shown. β-Actin and Tubulin were utilized as internal loading control. Data are represented as mean + SD of at least three independent experiments. Asterisks indicate the level of significance: * p<0.05, ** p<0.01, *** p<0.001 compared with the control cells (CTR).

Figure 8

Analysis of cell migration by scratch assay in melanoma cell lines after LY500307 treatment. Representative time-lapse microscopy images (upper panel) of wound closure of (A) Me1402R (B) Me665/1 and A375M (C) cells, untreated and treated with LY500307 (2μM and 4μM) for 24 hours. Bar graph of corresponding relative wounding area (lower panel). Results represent the mean + SD of at least four measurements of each wounded area, obtained in three independent experiments. Asterisks indicate the level of significance: * p<0.05, ** p<0.01, *** p<0.001 compared with the control cells (CTR).

Figure 9

Effect of LY500307 treatment on EMT-TFs. Representative WB analysis of TWIST and SLUG in Me665/1 (A), A375M (B) and Me1402/R (C) after 24h of treatment with increasing doses of LY500307 (2μM, 4μM, 8μM). E-cadherin expression analysis by WB (D) and flow cytometry (E) (CTR vs 8μM LY500307) in Me1402/R cells treated for 24 hours with LY500307. A representative FCM experiment out of three is shown. β-Actin or Tubulin were utilized as internal loading control. Densitometric quantifications shown as fold increase are represented as mean + SD of three independent experiments. Asterisks indicate the level of significance: * p<0.05, ** p<0.01 compared with the control cells (CTR).