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.

Keywords: ACC cell lines; ACC primary cells; adrenocortical carcinoma; estrogen receptors; progesterone receptors; tamoxifen.

Conflict of interest statement

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Copyright © 2021 Rossini, Tamburello, Abate, Beretta, Fragni, Cominelli, Cosentini, Hantel, Bono, Grisanti, Poliani, Tiberio, Memo, Sigala and Berruti.

Figures

Figure 1

ER expression in NCI-H295R, MUC-1 cell lines and ACC115m primary culture. Cells were seeded on poly-L-lysine pre-treated coverslips following by incubation with DAPI for nuclear staining. Panel (A) DAPI; panel (B) phalloidin; panel (C) ER (red signal: ER-α; green signal: ER-β); panel (D) merge. The scale bar of 20 µm is automatically inserted by the software ZEN Black.

Figure 2

Effect of tamoxifen on NCI-H295R cell viability and proliferation. (A) NCI-H295R were treated with increasing concentration of tamoxifen (0.1–20 uM) and cell viability was then evaluated by MTT assay. Results are expressed as percent of viable cells vs ctrl ± SEM of three independent experiments run in triplicate. (B) NCI-H295R were treated with low, intermediate, and high dose of tamoxifen and then cell proliferation was evaluated by directing counting with trypan blue discrimination. *P < 0.0001 vs untreated cells; §P < 0.001 vs untreated cells.

Figure 3

Tamoxifen exposure selectively modified the ER intracellular localization in NCI-H295R cells. (A) Cells were treated for different times with tamoxifen IC50 value. Slides were observed by a LSM 880 Zeiss confocal laser microscope or by a LSM 510 Zeiss confocal laser microscope (Carl Zeiss with 40× magnification. Images were then reconstructed using Zeiss ZEN 2.3 Imaging Software (Carl Zeiss). On the left the ER-β staining, on the right ER-β + DAPI staining. (B) The specific mean fluorescence intensity of the pixels of acquired images was quantified using ZEN Black software (Carl Zeiss). Several fields, randomly chosen, were acquired and then analyzed for each experimental condition. Quantified analysis was conducted by GraphPad Prism 5.02 software. *P < 0.0001 vs ctrl; #P < 0.01 vs ctrl.

Figure 4

PgR expression in NCI-H295R, MUC-1 cell lines and ACC115m primary culture. Cells were seeded on poly-L-lysine pre-treated coverslips following by incubation with DAPI for nuclear staining. Panel (A) DAPI; panel (B) phalloidin; panel (C): PgR; panel (D): merge. The scale bar of 20 µm is automatically inserted by the software ZEN Black.

Figure 5

Cytotoxic effect of Pg in ACC cell models. (A) MUC-1 cell line and ACC115m primary culture were treated with increasing concentrations of progesterone (0.1–160 uM), then cell viability was analyzed by MTT assay, (B) NCI-H295R, MUC-1 cell lines and ACC115m primary culture were treated with low, intermediate, and high dose of Pg, and cell proliferation was analyzed by directing counting with trypan blue discrimination. Results are expressed as percent of viable cells vs ctrl ± SEM; *P < 0.0001 vs untreated cells; #P < 0.01 vs untreated cells.

Figure 6

Combined treatment tamoxifen plus Pg in NCI-H295R. (A) Concentration–response curve of tamoxifen, Pg, and drug combination in NCI-H295R. Cells were exposed to increasing concentrations of tamoxifen and Pg alone or in combination as described in Materials and Methods. Data are expressed as percent of viable cells vs ctrl. Data are the mean ± SEM of three independent experiments; *P < 0.0001 vs untreated cells; §P < 0.001 vs untreated cells. (B) Combination index plot. Cell viability data of panel A were converted to Fa values and analyzed with CompuSyn software.

Figure 7

Tamoxifen and Pg reduced the SF-1 expression in NCI-H295R cell line. (1A) Representative western blot of SF-1 expression after NCI-H295R tamoxifen IC50 and Pg IC50 4 days treatment. (1B) Densitometric analysis of SF-1 expression after NCI-H295R drug treatment. Data are expressed as normalized values SF-1/GAPDH and are the mean of three independent experiments. *P < 0.0001 vs ctrl; §P < 0.001 vs ctrl. (1C) NCI-H295R were treated with tamoxifen IC50 or Pg IC50 for 4 days and then miRNA23a/b expression was investigated. Data are expressed as normalized values on internal control U6 and are the mean of three independent experiments. *P < 0.0001 vs ctrl; §P < 0.001 vs ctrl; #P < 0.01 vs ctrl. (2A) Representative western blot of SF-1 expression after MUC-1 tamoxifen IC50 and Pg IC50 5 days treatment. (2B) Densitometric analysis of SF-1 expression after MUC-1 drug treatment. Data are expressed as normalized values SF-1/GAPDH and are the mean of three independent experiments.

Figure 8

Immunohistochemistry for PgR and ER expression in ACC samples. Left panels show representative H&E-stained section from ACC tumor samples, middle panels PgR and right panels ER immunostainings. All images are from ×40 original magnification.