Ribociclib Cytotoxicity Alone or Combined With Progesterone and/or Mitotane in in Vitro Adrenocortical Carcinoma Cells

Abstract

Mitotane is the only approved drug for the treatment of adrenocortical carcinoma (ACC). The regimen to be added to mitotane is a chemotherapy with etoposide, doxorubicin, and cisplatin. This pharmacological approach, however, has a limited efficacy and significant toxicity. Target-therapy agents represent a new promising approach to cancer therapy. Among these, a preeminent role is played by agents that interfere with cell cycle progression, such as CDK4/6-inhibitors. Here, we investigated whether ribociclib could induce a cytotoxic effect both in ACC cell line and patient-derived primary cell cultures, alone or in combined settings. Cell viability was determined by MTT assay while cell proliferation was evaluated by direct count. Binary combination experiments were performed using Chou and Talalay method. Gene expression was analyzed by qRT-PCR while protein expression was evaluated by immunofluorescence. A double staining assay revealed that ribociclib induced a prevalent apoptotic cell death. Cell cycle analysis was performed to evaluate the effect of ribociclib treatment on cell cycle progression in ACC cell models. Our results indicated that ribociclib was cytotoxic and reduced the cell proliferation rate. The effect on cell viability was enhanced when ribociclib was combined with progesterone and/or mitotane. The effect of ribociclib on cell cycle progression revealed a drug-induced cell accumulation in G2 phase. The positive relationship underlined by our results between ribociclib, progesterone and mitotane strengthen the clinical potential of this combination.

An update on adrenocortical cell lines of human origin

“…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. Indeed, we demonstrated that progesterone receptors may be a druggable target in these ACC cells [35,36], thus confirming the correlation between the level of the progesterone receptor expression and the sensitivity of ACC cells to the cytotoxic effect elicited by progesterone [35,36,50,51]. Finally, the Cdk4/6 inhibitor ribociclib induces cytotoxicity and reduction of the cell proliferation rate in these cells, although the cell cycle perturbation appears to differ compared to other ACC cell lines.…”

“…Finally, the Cdk4/6 inhibitor ribociclib induces cytotoxicity and reduction of the cell proliferation rate in these cells, although the cell cycle perturbation appears to differ compared to other ACC cell lines. TVBF-7 cells accumulate, as expected, in G1 phase, while NCI-H295R cells and MUC-1 cells accumulate in G2 phase [36], thus supporting the experimental advantage to have different cell models that could reflect the different ACC behavior, both in vitro and in vivo. Interestingly, in line with the shared metastatic origin from patients in progression after EDP-M, the TVBF-7 cells reflect some, but not all, characteristics with the cell line MUC-1.…”

“…Interestingly, in line with the shared metastatic origin from patients in progression after EDP-M, the TVBF-7 cells reflect some, but not all, characteristics with the cell line MUC-1. In a recent published work, the exposure of these both cell models to mitotane, to progesterone and to the CDK4/6 inhibitor ribociclib alone or in combined setting, revealed a lower sensitivity compared to the NCI-H295R cells [36]. The different responses to pharmacological treatments underline the importance of their different phenotype.…”

“…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].…”