Mitotane (MTT) is an adrenolytic drug most used in the adjuvant and advanced treatment of adrenocortical carcinoma (ACC). Radiotherapy has so far had a limited use in the treatment of this cancer but recently its use has been reconsidered in this setting. The purpose of this study will be to investigate the effect of MTT and ionizing radiations (IR) singularly and in combination with them on cell growth inhibition and cell cycle perturbation in a xenograft model of ACC.
This study will be performed in H295R cell lines and in nude mice. Nude mice will be infected with H295R cells monitoring the neoplastic growth. After tumour development, animals will be stratified into groups including not infected mice, ACC infected mice to underwent different treatments.
The development of treated and untreated tumours will be monitored by MRI analyses. After mice sacrifice, the tumours will be removed to analysing cell cycle phases and DNA synthesis by FACScan cytofluorimeter. Molecules involved in the cell cycle will be investigated by Western blot analysis. However, will be assess the steroid levels in mice treated. Moreover, inducing a mishmatch repair protein (MMR) MLH1 and MSH2 downregulation in H295R cells will be study the role of this class of enzyme in treatment used.
MTT and IR in combination therapy represents a convincing new strategy to contrast the ACC. Our previous in vitro data has shown the capability of IR to increase the MTT effect when administered in combination with them (Cerquetti et al, ERC '08). In particular radiation therapy, in H295R and SW13 adrenocortical cell lines, induced a block in G2 phase of cell cycle which became irreversible when MTT was added in combination. This effect was accompanied by an increase of cdc2 high kinase activity and an up-regulation of cyclin B1 (Cerquetti et al, ERC '08). In addition, we demonstrated such as the cyclin B1 and cdk2 complex were strong involved in this biological processes as well. In fact, the administration of purvalanol A, a selective cdk-1 inhibitors, was able to overcame the cell cycle delay inducing apoptosis (Cerquetti et al, Int J Oncol '10). In the purposed study, as the primary objective, we will focus on the effect of the MTT/IR in vivo individually and in combination with them in an ACC xenograft model. Moreover, we previously provided evidence about the radiosensitizing property of MTT, attributing it to a reduction of expression level of hMLH1 and hMSH2, belonging to the family of mishmatch repair protein (MMR) (Cerquetti et al, Int J Oncol '10). The MMR system, a family of proteins involved in repair of base-base mismatches and/or other type of mutations occurring during DNA replication, plays a crucial role in many biological process such as the response to injury induced by chemical and physical agents (Martin SA et al, Clin Canc Res'10). Recently, the idea of reducing or losing the MMR function could be a promising strategy for clinical oncology (Martin SA et al, Clin Canc Res'10). MTT is known as steroidogenesis inhibitor and our previous data in vitro demonstrated as the combination therapy was able to reduce the cortisol level more than the MTT and IR alone (Cerquetti et al, ERC '08).
In view of these remarks we believe that both treatments in combination with them can represent the overcoming of the limits of those treatments by themselves either in terms of hormonal control or the antiproliferative effect in therapeutic strategy of ACC. This effect could improve the compliance of the patients affected by clinical shapes of functioning ACC.
Fassnacht et al. (JCEM '06) already identified an opportunity in radiotherapy treatment in a retrospective study in patients affected by ACC. Polat et al. (Cancer '09) reinforced this hypothesis, describing in a review the experience of their center, indicating the potential therapeutic protocols. Finally, Sabolch et al. (Int J Rad Oncol Biol Phys '11) in a first report describe the effectiveness of radiotherapy in a large cohort of patients affected by ACC. This data regard to use of radiotherapy alone after adrenalectomy in patients affected by ACC at different stages. Another more recent paper from the same group, demonstrated a significant postoperative improvement of local control in a retrospective series of 20 patients underwent Ro or R1 resection followed by adjuvant radiotherapy (Sabolch A, Int J Rad Oncol Biol Phys '15).
These findings open the perspective of a rationale employment of combination of a drug classically used in ACC therapy with IR in this neoplasia.
However we believe that the data emerging from the literature about the potential offers of radiotherapy in the ACC treatment with those previously produced by us can represent an important requirements for the development of future clinical strategies to counteract the ACC. In particular this study is proposed as a preclinical model of a new therapeutic regimen.