XB-ART-53345
Dis Model Mech
2015 Oct 01;810:1237-46. doi: 10.1242/dmm.021071.
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A calixpyrrole derivative acts as an antagonist to GPER, a G-protein coupled receptor: mechanisms and models.
Lappano R
,
Rosano C
,
Pisano A
,
Santolla MF
,
De Francesco EM
,
De Marco P
,
Dolce V
,
Ponassi M
,
Felli L
,
Cafeo G
,
Kohnke FH
,
Abonante S
,
Maggiolini M
.
Abstract
Estrogens regulate numerous pathophysiological processes, mainly by binding to and activating estrogen receptor (ER)α and ERβ. Increasing amounts of evidence have recently demonstrated that G-protein coupled receptor 30 (GPR30; also known as GPER) is also involved in diverse biological responses to estrogens both in normal and cancer cells. The classical ER and GPER share several features, including the ability to bind to identical compounds; nevertheless, some ligands exhibit opposed activity through these receptors. It is worth noting that, owing to the availability of selective agonists and antagonists of GPER for research, certain differential roles elicited by GPER compared with ER have been identified. Here, we provide evidence on the molecular mechanisms through which a calixpyrrole derivative acts as a GPER antagonist in different model systems, such as breast tumor cells and cancer-associated fibroblasts (CAFs) obtained from breast cancer patients. Our data might open new perspectives toward the development of a further class of selective GPER ligands in order to better dissect the role exerted by this receptor in different pathophysiological conditions. Moreover, calixpyrrole derivatives could be considered in future anticancer strategies targeting GPER in cancer cells.
PubMed ID: 26183213
PMC ID: PMC4610237
Article link: Dis Model Mech
Species referenced: Xenopus
Genes referenced: akt1 ccn1 ccn2 egr1 fos mapk1 tbx1 tff3.1
Article Images: [+] show captions
Fig. 1. Chemical structure of meso-octamethylcalix[4]pyrrole (C4PY). | |
Fig. 2. Ligand binding modes to GPER. (A) C4PY in the protein binding cleft is drawn in green. The protein surface is colored according to its electrostatic potential (blue positive, red negative). The same ligand binding mode is schematically reported in panel B, where the interacting amino acids are indicated as dark gray sticks. (C,D) The agonists GPER-L1 and GPER-L2 are drawn in light green (C) and purple (D) sticks, respectively. Binding mode of G-1 (cyan) is shown in panel E and the full-antagonist MIBE (orange) in panel F. | |
Fig. 3. C4PY is a ligand of GPER. (A) C4PY competes with [3H]E2 for binding to GPER in SkBr3 cells. Competition curves of increasing concentration of unlabeled E2, G-1 and C4PY expressed as a percentage of maximum specific [3H]E2 binding. Each data point represents the mean±s.d. of triplicate samples of three separate experiments. (B) C4PY competes with [5,6-3H] nicotinic acid (NA) for binding to GPER in SkBr3 cells. Competition curves of increasing concentration of unlabeled NA, G-1 and C4PY expressed as a percentage of maximum specific [5,6-3H] NA binding. Each data point represents the mean±s.d. of three separate experiments performed in triplicate. | |
Fig. 4. C4PY exerts inhibitory effects through GPER in SkBr3 breast cancer cells. (A) ERK1/2 and Akt activation in SkBr3 cells treated for 15â min with 100â nM E2 or 1â µM G-1 is prevented in the presence of 1â µM C4PY. (B) Densitometric analysis of the blots normalized to ERK2 and Akt, respectively. Each data point represents the mean±s.d. of three independent experiments. (C) The mRNA expression of fos and EGR1 induced in SkBr3 cells by 1â h treatment with 100â nM E2 and 1â µM G-1 is inhibited in the presence of 1â µM C4PY, as evaluated by real-time PCR. Results obtained from experiments performed in triplicate were normalized for 18S expression and shown as fold change of RNA expression compared to cells treated with vehicle. Each data point represents the mean±s.d. of three independent experiments performed in triplicate. (D) The transactivation of fos and EGR1 luciferase reporter genes transfected in SkBr3 cells induced by 100â nM E2 and 1â µM G-1 is inhibited by 1â µM C4PY. Luciferase activity was normalized to the internal transfection control Renilla luciferase; values are presented as fold change (mean±s.d.) of vehicle control and represent three independent experiments, each performed in triplicate. (E) The proliferation of SkBr3 cells upon treatment with 100â nM E2 and 100â nM G-1 is inhibited by 1â µM C4PY, as indicated. Cells were treated for 5â days with the indicated treatments and counted on day 6. Proliferation of cells receiving vehicle was set as 100%, upon which cell growth induced by treatments was calculated. Each data point is the average ±s.d. of three independent experiments performed in triplicate. (â¢) indicates P<0.05 for cells receiving vehicle (â) versus treatments. | |
Fig. 5. C4PY exerts inhibitory effects through GPER in CAFs. (A) ERK1/2 and Akt activation in CAFs treated for 5â min with 1â nM E2 and 100â nM G-1 is prevented by 1â µM C4PY. (B) Densitometric analysis of the blots normalized to ERK2 and Akt, respectively. Each data point represents the mean±s.d. of three independent experiments. (C) The mRNA expression of CTGF and Cyr61 induced in CAFs by 1â h treatment with 1â nM E2 and 100â nM G-1 is prevented by 1â µM C4PY, as evaluated by real-time PCR. Results obtained from experiments performed in triplicate were normalized for 18S expression and shown as fold change of RNA expression compared to cells treated with vehicle. Each data point represents the mean±s.d. of three independent experiments performed in triplicate. (D) CTGF and Cyr61 protein expression induced in CAFs by 2â h treatment with 1â nM E2 and 100â nM G-1 is inhibited in the presence of 1â µM C4PY. (E) Densitometric analyses of the blots normalized to β-actin; values shown represent the mean±s.d. of three independent experiments. (F) The migration of CAFs upon treatment with 1â nM E2 and 100â nM G-1 is inhibited by 1â µM C4PY, as evaluated by Boyden Chamber assay. Each data point is the average ±s.d. of three independent experiments performed in triplicate. (â¢) and (â¦) indicate P<0.05 for cells receiving vehicle (â) versus treatments. | |
Fig. 6. The migration of CAFs induced by E2 (1â nM) and G-1 (100â nM) is inhibited by 1â µM C4PY, as determined by wound-healing assay. Data are representative of three independent experiments performed in triplicate. | |
Fig. 7. C4PY does not interfere with the ER-mediated signaling. (A) MCF-7 cells were transfected with an ER luciferase reporter gene along with the internal transfection control Renilla luciferase and then treated with 10â nM E2 in combination with 1â µM C4PY or ICI, as indicated. The normalized luciferase activity values of cells treated with vehicle were set as 1-fold induction, upon which the activity induced by treatments was calculated. Each data point represents the mean±s.d. of three experiments performed in triplicate. (B) The mRNA expression of cyclin D1 (Cyc D1), progesterone receptor (PR) and pS2 induced in MCF-7 cells by 24â h treatment with 10â nM E2 is inhibited by 1â µM ICI, but not by 1â µM C4PY, as evaluated by real-time PCR. Results obtained from experiments performed in triplicate were normalized for 18S expression and shown as fold change of RNA expression compared to cells treated with vehicle. Each data point represents the mean±s.d. of three independent experiments performed in triplicate. (C) The proliferation of MCF-7 cells upon treatment with 10â nM E2 is inhibited by 1â µM ICI, but not by 1â µM C4PY, as indicated. Cells were treated for 5â days with the indicated treatments and counted on day 6. Proliferation of cells receiving vehicle was set as 100%, upon which cell growth induced by treatments was calculated. Each data point is the average ±s.d. of three independent experiments performed in triplicate. (â¢) indicates P<0.05 for cells receiving vehicle (â) versus treatments. |
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