Estrogens Modulate Somatostatin Receptors Expression and Synergize With the Somatostatin Analog Pasireotide in Prostate Cells

Abstract

Prostate cancer (PC) is one of the most frequently diagnosed cancers and a leading cause of cancer-related deaths in Western society. Current PC therapies prevalently target the functions of androgen receptor (AR) and may only be effective within short time periods, beyond which the majority of PC patients progress to castration-resistant PC (CRPC) and metastatic disease. The role of estradiol/estradiol receptor (ER) axis in prostate transformation and PC progression is well established. Further, considerable efforts have been made to investigate the mechanism by which somatostatin (SST) and somatostatin receptors (SSTRs) influence PC growth and progression. A number of therapeutic strategies, such as the combination of SST analogs with other drugs, show, indeed, strong promise. However, the effect of the combined treatment of SST analogs and estradiol on proliferation, epithelial mesenchyme transition (EMT) and migration of normal- and cancer-derived prostate cells has not been investigated so far. We now report that estradiol plays anti-proliferative and pro-apoptotic effect in non-transformed EPN prostate cells, which express both ERα and ERβ. A weak apoptotic effect is observed in transformed CPEC cells that only express low levels of ERβ. Estradiol increases, mainly through ERα activation, the expression of SSTRs in EPN, but not CPEC cells. As such, the hormone enhances the anti-proliferative effect of the SST analog, pasireotide in EPN, but not CPEC cells. Estradiol does not induce EMT and the motility of EPN cells, while it promotes EMT and migration of CPEC cells. Addition of pasireotide does not significantly modify these responses. Altogether, our results suggest that pasireotide may be used, alone or in combination with other drugs, to limit the growth of prostate proliferative diseases, provided that both ER isoforms (α and β) are present. Further investigations are needed to better define the cross talk between estrogens and SSTRs as well as its role in PC.

Keywords: EMT; apoptosis; estrogens; migration; prostate cancer; somatostatin analogs; somatostatin receptors.

FIGURE 1

Effect of estradiol on SSTRs mRNA and protein expression levels. RT-PCR analysis of SSTR1, 2, 3 and 5 mRNA levels in EPN (A) or CPEC (B) cells untreated or treated for 24h with 20 nM estradiol (E₂) was done. In panel (A,B), densitometry analysis of agarose gel electrophoresis is shown. The expression level is indicated as fold changes over the basal conditions (A.U.). Histograms represent the averages (+/– standard error) from 3 independent experiments, normalized for the expression of the control housekeeping gene GAPDH (^∗indicates p < 0.05 for each gene versus its untreated control). Means and SEMs are shown. n represents the number of experiments. ^∗p < 0,05. Insets are representative from one experiment in A or B. In panel (C, upper section), the Western blot analysis of lysate proteins from EPN and CPEC cell lines untreated or treated with estradiol (20 nM; E₂) for 48 h was done. Lysate proteins (1 mg/ml) were resolved by SDS–PAGE, transferred to PVDF membrane and probed with anti-SSTRs or anti-tubulin antibodies. Inset in panel (C), densitometry analysis of SSTRs was done in two different experiments using the NIH/Image J software. Results were expressed as relative change over the basal level of each SSTR isoform. In panel (C, lower section), the Western blot analysis of lysate proteins from EPN cells untreated or treated with estradiol (20 nM; E₂) or PPT (3 nM) or DPN (3 nM) for 48 h was done. Lysate proteins (2 mg/ml) were resolved by SDS–PAGE, transferred to nitrocellulose membrane and probed with the antibodies against the indicated proteins. In panel (C, upper and lower sections), the Western blot analysis of tubulin is shown, as loading control.

FIGURE 2

Effect of estradiol on cell cycle. Flow cytometry analysis of propidium iodide-labeled EPN (A) or CPEC (B) cells was done, as described in Methods. Quiescent cells were left under basal conditions or treated with estradiol (20 nM; E₂) for the indicated times. Histograms represent the % counts/FL2 areas of labeled cells in the different cell cycle phases after 24 or 48 h of hormonal treatment. Means and SEMs are shown. n represents the number of experiments. ^∗p < 0,05 for each experimental point vs. the corresponding untreated control.

FIGURE 3

Effect of both estradiol and pasireotide on cell cycle. Flow cytometry analysis of propidium iodide-labelled EPN (A) or CPEC (B) was done, as described in Methods. In panel (A,B), quiescent cells were left under untreated or treated for 24 h with 0,1 μM pasireotide, in the absence or presence of 20 nM estradiol. Histograms in panel (A,B) represent the % counts/FL2 areas of labeled cells in the different cell cycle phases after the indicated treatments. Means and SEMs are shown. n represents the number of experiments. ^∗p < 0,05; ^∗∗p < 0,01. In panel (C), quiescent EPN (left section) or CPEC (right section) cells were left untreated or treated for 24 h with 0,1 μM pasireotide, in the absence or presence of 20 nM estradiol. Lysate proteins were separated by SDS-PAGE, transferred to PDVF membrane and filters were then analyzed by Western blot, using the anti-activated caspase 3 or anti-tubulin antibodies.

FIGURE 4

Effect of both estradiol and pasireotide on Bcl-2 and Myc mRNA expression levels. EPN or CPEC cells were untreated or treated for 24 h with estradiol (20 nM; E₂), in the absence or presence of 1 μM pasireotide. RT-PCR analysis of Bcl-2 (A) and c-Myc (B) mRNA levels was done. Densitometry analysis of agarose gel electrophoresis was done. The expression level is indicated as fold changes from basal conditions (A.U.). Histograms represent the averages (+/- standard error) from five independent experiments, normalized for the expression of the control housekeeping gene GAPDH. Means and SEMs are shown. n represents the number of experiments. ^∗p < 0.05 for each experimental point versus the corresponding untreated control. In panel (C), EPN or CPEC cells were untreated or treated for 24 h with estradiol (20 nM; E₂), in the absence or presence of 1 μM pasireotide. Lysate proteins were prepared, electrophoretically separated by SDS-PAGE and transferred to PDVF membrane. Filters were immune-blotted using anti-Bcl-2 or anti-c Myc or anti-tubulin antibodies.

FIGURE 5

Effect of estradiol and pasireotide on EMT, morphology and motility of EPN cells. Quiescent EPN cells were untreated or treated for 48 h with the indicated compounds. Estradiol was used at 20 nM, pasireotide at 0.1 mM, PPT and DPN both at 3 nM. In panel (A), lysate proteins (2 mg/ml) were prepared, separated by SDS-PAGE and transferred to nitrocellulose membrane. Filters were immune-blotted using the antibodies against the indicated proteins. The blots are representative of two different experiments. In panel (B), the cells were analyzed for morphological changes using contrast-phase microscopy. Bar, 10 mM. In panel (C), the cells were wounded and then left unstimulated or stimulated with the indicated compounds. Cytosine arabinoside (20 mM) was added to the cell medium to avoid cell proliferation. Contrast-phase images in panel (B,C) are representative of 3 different experiments.

FIGURE 6

Effect of estradiol and pasireotide on EMT, morphology and motility of CPEC cells. Quiescent CPEC cells were untreated or treated for 48 h with the indicated compounds. Estradiol was used at 20 nM, pasireotide at 0,1 μM and DPN at 3 nM. In panel (A), lysate proteins (2 mg/ml) were prepared, separated by SDS-PAGE and transferred to nitrocellulose membrane. Filters were immune-blotted using the antibodies against the indicated proteins. The blots are representative of two different experiments. In panel (B), the cells were analyzed for morphological changes using contrast-phase microscopy. Bar, 10 μM. In panel (C), the cells were wounded (the arrow indicates the wound length) and then left unstimulated or stimulated with the indicated compounds. Cytosine arabinoside (20 μM) was added to the cell medium to avoid cell proliferation. The wound gap from three different experiments was calculated using Image J software and expressed as % of the decrease in wound area. Means and SEM are shown. n represents the number of experiments. ^∗p < 0.05 for each experimental point versus the corresponding untreated control. Contrast-phase images in panel (D) are representative of 3 different experiments. In panel (C,D), quiescent cells on coverslips were left unstimulated or stimulated for 24 h with estradiol (20 nM), or DPN (3 nM), or pasireotide (0,1 μM) or a combination of estradiol plus pasireotide. After in vivo pulse with BrdU (100 μM; Sigma), the DNA synthesis was analyzed by IF as reported in Methods and calculated by the formula: percentage of BrdU-positive cells = (No. of BrdU-positive cells/No. of total cells) X 100. In unstimulated cells, the BrdU incorporation was observed in less than 14% of total cells. Neither estradiol, nor DPN, nor pasireotide, nor combination of estradiol and pasireotide increased the BrdU incorporation, which was detected in 18, 16, 15, and 17% of total cells, respectively.