We have previously demonstrated that estrogen receptor (ER) alpha (ESR1) increases proliferation of adrenocortical carcinoma (ACC) through both an estrogen-dependent and -independent (induced by IGF-II/IGF1R pathways) manner. Then, the use of tamoxifen, a selective estrogen receptor modulator (SERM), appears effective in reducing ACC growth in vitro and in vivo. However, tamoxifen not only exerts antiestrogenic activity, but also acts as full agonist on the G protein-coupled estrogen receptor (GPER). Aim of this study was to investigate the effect of a non-steroidal GPER agonist G-1 in modulating ACC cell growth. We found that G-1 is able to exert a growth inhibitory effect on H295R cells both in vitro and, as xenograft model, in vivo. Treatment of H295R cells with G-1 induced cell cycle arrest, DNA damage and cell death by the activation of the intrinsic apoptotic mechanism. These events required sustained extracellular regulated kinase (ERK) 1/2 activation. Silencing of GPER by a specific shRNA partially reversed G-1-mediated cell growth inhibition without affecting ERK activation. These data suggest the existence of G-1 activated but GPER-independent effects that remain to be clarified. In conclusion, this study provides a rational to further study G-1 mechanism of action in order to include this drug as a treatment option to the limited therapy of ACC.

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.

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Purpose: Melanoma patients have a very poor prognosis with a response rate of <1% due to advanced diagnosis. This type of tumor is particularly resistant to conventional chemotherapy and radiotherapy, and the surgery remains the principal treatment for patients with localized melanoma. For this reason, there is particular interest in the melanoma biological therapy. Experimental Design: Using two p53 mutant melanoma models stably expressing an inducible c-myc antisense RNA, we have investigated whether Myc protein down-regulation could render melanoma cells more susceptible to radiotherapy, reestablishing apoptotic p53-independent pathway. In addition to address the role of p53 in the activation of apoptosis, we studied the effect of Myc down-regulation on radiotherapy sensitivity also in a p53 wild-type melanoma cell line. Results: Myc down-regulation is able per se to induce apoptosis in a fraction of the cell population (f40% at 72 hours) and in combination with g radiation efficiently enhances the death process. In fact, f80% of apoptotic cells are evident in Myc down-regulated cells exposed to g radiation for 72 hours compared with f13% observed after only g radiation treatment. Consistent with the enhanced apoptosis is the inhibition of the MLH1 and MSH2 mismatch repair proteins, which, preventing the correction of ionizing radiation mismatches occurring during DNA replication, renders the cells more prone to radiation-induced apoptosis. Conclusions: Data herein reported show that Myc down-regulation lowers the apoptotic threshold in melanoma cells by inhibiting MLH1and MSH2 proteins, thus increasing cell sensitivity to g radiation in a p53-independent fashion. Our results indicate the basis for developing new antitumoral therapeutic strategy, improving the management of melanoma patients.

Infertility is a dramatic and frequent side effect in women who are undergoing chemotherapy. Actual strategies are mainly focused on oocyte cryopreservation, but this is not always a suitable option. Considering the key role that granulosa cells play in follicle life, we studied whether thyroid hormone 3,5,3 0 -triiodothyronine (T 3 ) protects rat ovarian granulosa cells from chemotherapy-induced apoptosis. To this aim, a cell line was established from fresh isolated rat granulosa cells and named rGROV. Cells were exposed to paclitaxel (PTX) and T 3 , and apoptosis, cell viability, and cell cycle distribution were analyzed under different conditions. First, the integrity of the steroidogenic pathway was demonstrated, and the presence of thyroid receptors, transporters, and deiodinases was confirmed by quantitative PCR. Cells were then exposed to PTX alone or contemporary to T 3 . MTT and TUNEL assays revealed that while there was a relevant percentage of dying cells when exposed to PTX (40-60%), the percentage was sensibly reduced (20-30%) in favor of living cells if T 3 was present. Cell cycle analysis showed that cells exposed to PTX alone were first collected in G2 and then died by apoptosis; on the other hand, the T 3 granted the cells to cycle regularly and survive PTX insult. In addition, western blot and FCM analyses confirmed that caspases activation, casp 3 and Bax, were downregulated by T 3 and that Bcl2 and cyclins A and B together with cdk1 were upregulated by T 3 . In conclusion, we demonstrated that thyroid hormone T 3 can counteract the lethal effect of taxol on granulosa cells.