Mitotane Induce Difference Modes of Cell Death in Treatment Resistant and Sensitive Models of Adrenocortical Carcinoma
Sarah Feely (1), Nathan Mullen (1), Cong Hong (1), Abhay Pandit (2), Constanze Hantel (3), William Rainey (4), Michael Conall Dennedy (1)
1. Discipline of Pharmacology and Therapeutics, School of Medicine, University of Galway, Ireland. 2. Science Foundation Ireland (SFI) Centre for Research in Medical Devices (CURAM), Biomedical Science Building, University of Galway, Ireland 3. Department of Medicine IV, University Hospital, Ludwig Maximilian University of Munich, Munich, Germany. 4. Department of Molecular and Integrative Physiology, University of Michigan, Ann Arbor, Michigan
Adrenocortical carcinoma (ACC) is a rare aggressive cancer with poor survival. Adjuvant mitotane is the only approved drug for treatment of ACC. It improves survival but its use is limited by poor tolerability and drug resistance. For metastatic ACC, combination chemotherapy, using mitotane and etoposide, doxorubicin, and cisplatin improves survival, but efficacy is limited. Better understating the cytotoxic mechanisms of mitotane offers potential to develop improved therapeutic options for ACC.
To investigate the underlying mechanisms in vitro, human ACC cells (HAC15 and H295R), in monolayer cell culture were treated with increasing concentrations of mitotane. In order to determine the sensitivity of these cells to apoptosis and necroptosis, all cells were treated with varying concentrations of staurosporine and TNF-alpha respectively. Cell death was evaluated at 24 hours using flow cytometry analysis of Annexin V and Sytox Blue. This was complemented by Incucyte ® Live-Cell Analysis System imaging of annexin V and Sytox Blue staining. Expression of the markers of apoptosis, cleaved caspase 3 , and necroptosis, phosphorylated MLKL, RIP3 and RIP1 was measured at 24 hours.
Mitotane significantly reduced the viability of H295R cells at 24 hours at the therapeutic concentration of 50uM (p>0.0001), thereby demonstrating treatment-sensitivity. In HAC15 cells, a supratherapeutic dose of 200uM was needed to significantly reduce viability (p>0.05) at 24 hours, indicating resistance to mitotane treatment at therapeutic concentrations. There was increasing Annexin V fluorescence with increasing mitotane concentration in H295R and HAC15 cells at 24 hours without higher coinciding staining with Sytox Blue. Video analysis demonstrated these findings. Cleaved caspase 3 expression was associated with mitotane-induced cell death in H295R cells at 24 hours, indicating apoptosis. In HAC15 cells, there was no appreciable expression of cleaved caspase 3 at24 hours. Phosphorylated-MLKL and RIP1 expression was seen in controls and all mitotane doses in both cell lines at both timepoints, indicating necroptosis. Phosphorylated RIP3 expression was only seen in HAC15 cells.
In this study, H295R cells, representing a treatment-sensitive model of Mitotane undergo both apoptotic and necroptotic cell death at 24 hours of mitotane exposure. In contrast, HAC15 cells, demonstrated relative treatment resistance to mitotane, and undergo necroptotic cell death only. Interestingly, necroptosis only was induced in these cells by staurosporine, an known inducer of apoptosis. These results highlight the importance of understanding and targeting non-apoptotic and necroptotic cell death pathways, particularly when evaluating mechanisms of treatment resistance in ACC. Given the difference in the mechanism of cell death in treatment-sensitive and treatment-resistant cells, further exploration of necroptosis is necessary to better understand how ACC cells may manifest treatment resistance.