Mitotane increases the radiotherapy inhibitory effect and induces G2-arrest in combined treatment on both H295R and SW13 adrenocortical cell lines

Abstract

Mitotane, 1,1-dichloro-2-(o-chlorophenyl)-2-(p-chlorophenyl)ethane (o,p 0 -DDD) is an agent with adrenotoxic effect, which is able to block cortisol synthesis. This drug and radiotherapy are used also in adrenal cancer treatment even if their biological action in this neoplasia remains unknown. We investigated the effects of o,p 0 -DDD and ionizing radiations (IR) on cell growth inhibition and cell cycle perturbation in H295R and SW13 adrenocortical cancer cells. Both cell lines were irradiated at a 6 Gy dose and were treated with o,p 0 -DDD 10 K5 M separately and with IR/o,p 0 -DDD in combination. This combination treatment induced an irreversible inhibition of cell growth in both adrenocortical cancer cells. Cell cycle analysis showed that IR alone and IR/o,p 0 -DDD in combination induced the cell accumulation in the G 2 phase. At 120 h after IR, the cells were able to recover the IR-induced G 2 block while cells treated with IR/o,p 0 -DDD were still arrested in G 2 phase. In order to study the molecular mechanism involved in the G 2 irreversible arrest, we have considered the H295R cell line showing the highest inhibition of cell proliferation associated with a noteworthy G 2 arrest. In these cells, cyclin B1 and Cdk2 proteins were examined by western blot and Cdk2 kinase activity measured by assay kit. The H295R cells treated with IR/o,p 0 -DDD shared an increase in cyclin B1 amount as the coimmunoprecipitation of Cdc2-cyclin B1 complex. The kinase activity also shows an increase in the treated cells with combination therapy. Moreover, in these cells, sequence analysis of p53 revealed a large deletion of exons 8 and 9. The same irreversible block on G 2 phase, induced by IR/o,p 0 -DDD treatment, happened in H295R cells with restored wild-type p53 suggesting that this mechanism is not mediated by p53 pathway.

Cytotoxic activity of gemcitabine, alone or in combination with mitotane, in adrenocortical carcinoma cell lines

“…However, this effect on cell proliferation was not paralleled by a significant influence on apoptosis and cell cycle in mitotane-sensitive H295R cells. This observation, already presented in previous reports detailing the effects of mitotane on cell cycle used as a single agent (Cerquetti et al, 2008), supports a cytostatic rather than cytotoxic effect of mitotane in ACC cells.…”

“…Second, the anti-neoplastic properties of mitotane seem to be adrenocortical-specific, since mitotane does not appear to be effective on other tumor cells lines, such as those of lung origin (Volante et al, 2012). Moreover, the mechanisms underlying its adrenocortical-specific anti-neoplastic properties are largely unknown and possibly related to different biological processes (including energetic metabolism, stress response and other cellular functions), as suggested by proteomic analysis of H295R cells (Stigliano et al, 2008). Finally, the possible interactions of mitotane with other antitumor agents, including the chemotherapeutic drugs commonly used for ACC treatment, are poorly explored.…”

“…Finally, the possible interactions of mitotane with other antitumor agents, including the chemotherapeutic drugs commonly used for ACC treatment, are poorly explored. In this latter respect, mitotane has been shown to sensitize ACC cancer cells to ionizing radiation (Cerquetti et al, 2008) and to enhance cytotoxicity of chemotherapy by reversing P-glycoprotein-mediated multidrug resistance with a specific effect on the cell cycle (Bates et al, 1991). However, very few studies determined in vitro the activity of mitotane in combination with other chemotherapeutic agents used in ACC patients (Villa et al, 1999).…”

Integrative molecular bioinformatics study of human adrenocortical tumors: microRNA, tissue-specific target prediction, and pathway analysis

“…CCNB/ CDC2 complex was found to be overexpressed in Endocrine-Related Cancer (2009) 16 895-906 www.endocrinology-journals.org several malignancies (Egloff et al 2006); however, its exact role in ACC is unclear. Treatment of ACC cell lines either with 17b-estradiol (E 2 ) and progesterone (Brown et al 2008) or with the adrenolytic compound mitotane combined with irradiation therapy (Cerquetti et al 2008) induced G2/M block and apoptosis. Progesterone alone and in combination with E 2 induced mitotic perturbations in SW-13 cells via decreased CCNB1 and CCND1 expression (Brown et al 2008).…”

Expression of cyclin B1 after induction of senescence and cell death in non-small cell lung carcinoma A549 cells

“…On the other hand, Dan and Yamori [15] suggested that changes in the level of cyclin B1 and G2/M arrest in A549 cells exposed to doxorubicin are regulated in the manner independent of p53, because similar changes were not induced by cisplatin, a well-known p53 activator [15]. In agreement with that, it was shown that an increased level of cyclin B1 may appear in some G2/M-arrested cancer cells with defects in p53 [16]. However, we could not exclude that the increase in the cyclin level observed by us may be strictly correlated with morphological changes appearing in this population of cells after treatment with doxorubicin.…”