Cytotoxic Effect of Progesterone, Tamoxifen and Their Combination in Experimental Cell Models of Human Adrenocortical Cancer

Abstract

Progesterone (Pg) and estrogen (E) receptors (PgRs and ERs) are expressed in normal and neoplastic adrenal cortex, but their role is not fully understood. In literature, Pg demonstrated cytotoxic activity on AdrenoCortical Carcinoma (ACC) cells, while tamoxifen is cytotoxic in NCI-H295R cells. Here, we demonstrated that in ACC cell models, ERs were expressed in NCI-H295R cells with a prevalence of ER-β over the ER-α.Metastasis-derived MUC-1 and ACC115m cells displayed a very weak ER-α/β signal, while PgR cells were expressed, although at low level. Accordingly, these latter were resistant to the SERM tamoxifen and scarcely sensitive to Pg, as we observed a lower potency compared to NCI-H295R cells in cytotoxicity (IC50: MUC-1 cells: 67.58 µM (95%CI: 63.22–73.04), ACC115m cells: 51.76 µM (95%CI: 46.45–57.67) and cell proliferation rate. Exposure of NCI-H295R cells to tamoxifen induced cytotoxicity (IC50: 5.43 µM (95%CI: 5.18–5.69 µM) mainly involving ER-β, as their nuclear localization increased after tamoxifen: Δ A.U. treated vs untreated: 12 h: +27.04% (p < 0.01); 24 h: +36.46% (p < 0.0001). This effect involved the SF-1 protein reduction: Pg: −36.34 ± 9.26%; tamoxifen: −46.25 ± 15.68% (p < 0.01). Finally, in a cohort of 36 ACC samples, immunohistochemistry showed undetectable/low level of ERs, while PgR demonstrated a higher expression. In conclusion, ACC experimental cell models expressed PgR and low levels of ER in line with data obtained in patient tissues, thus limiting the possibility of a clinical approach targeting ER. Interestingly, Pg exerted cytotoxicity also in metastatic ACC cells, although with low potency.

Innovative multidimensional models in a high-throughput-format for different cell types of endocrine origin

“…Cells from benign pheochromocytoma and benign adrenal hyperplasia were plated at 250000 cells /well in the spherical plate 5D (Kugelmeiers, Erlenbach, Switzerland). NCI-H295R, MUC-1 and ACC115m primary cells were cultured as previously described (Hantel et al, 2016; Rossini et al, 2021) and cultivated in Sphericalplates 5D for either 7 or 14 days at the densities of 75000 cells/well, 150000 cells/well and 75000 cells/well respectively. The 7 day spheroids were treated with 650nM Adavosertib (MedChemExpress) for 72 h. Additionally, ACC115m cells were cultivated in Sphericalplates 5D for 7 days and subsequently treated with 25μM Gemcitabine and 40μM Cisplatin (both Teva Pharma) for 72h.…”

“…Fortunately, this gap could recently been narrowed with cell models that are now applicable for a spectrum of research applications (Abate et al, 2020; Fragni et al, 2019; Hantel et al, 2016; Hasanovic et al, 2018; Landwehr et al, 2021; Liang et al, 2020; E. M. Pinto, Kiseljak-Vassiliades, & Hantel, 2019; Rossini et al, 2021; Siebert et al, 2019; Warde et al, 2020). However, there is still potential for improvement by translating adrenocortical cells towards 3D culture and appropriate personalized approaches in vitro.…”

An update on adrenocortical cell lines of human origin

“…The model shows the for metastases frequently observed increased cellular plasticity, which can be e.g. seen in highly elevated drug tolerance for a wide range of chemotherapeutic, phytochemical, molecular targeted drugs and also combinatory regimens which is far beyond resistance against the originally applied EDP-M scheme [10,[27][28][29][30][31][32][33][34][35][36]. Recently, a broad comparative drug screen of clinically relevant chemotherapies and targeted therapies has been performed in NCI-H295R and MUC-1.…”

“…Recently, the new ACC cell line TVBF-7 has been proposed by our group [13]. These cells represent the most recently developed ACC cell line, formerly known as primary cell culture ACC115m [35,36]. Cells were established from a perirenal lymph-node metastasis of a male ACC patient which, similar to MUC-1, underwent progression upon clinical EDP-M treatment [13].…”

“…Except for the ACTH receptor (MC2R), these cells express, compared to NCI-H295R, low level of hormonal receptor at gene level [13,35]. The expression of estrogen receptor α is comparable to that of NCI-H295R, while the expression of estrogen receptor β is lower [35]. TVBF-7 cells have already been used as an experimental cell model to test the efficacy of drugs proven effective in ACC, in addition to other ACC cell lines.…”