Cytotoxic Effect of Progesterone, Tamoxifen and Their Combination in Experimental Cell Models of Human Adrenocortical Cancer

Progesterone (Pg) and estrogen (E) receptors (PgRs and ERs) are expressed in normal and neoplastic adrenal cortex, but their role is not fully understood. In literature, Pg demonstrated cytotoxic activity on AdrenoCortical Carcinoma (ACC) cells, while tamoxifen is cytotoxic in NCI-H295R cells. Here, we demonstrated that in ACC cell models, ERs were expressed in NCI-H295R cells with a prevalence of ER-β over the ER-α.Metastasis-derived MUC-1 and ACC115m cells displayed a very weak ER-α/β signal, while PgR cells were expressed, although at low level. Accordingly, these latter were resistant to the SERM tamoxifen and scarcely sensitive to Pg, as we observed a lower potency compared to NCI-H295R cells in cytotoxicity (IC50: MUC-1 cells: 67.58 µM (95%CI: 63.22-73.04), ACC115m cells: 51.76 µM (95%CI: 46.45-57.67) and cell proliferation rate. Exposure of NCI-H295R cells to tamoxifen induced cytotoxicity (IC50: 5.43 µM (95%CI: 5.18-5.69 µM) mainly involving ER-β, as their nuclear localization increased after tamoxifen: Δ A.U. treated vs untreated: 12 h: +27.04% (p < 0.01); 24 h: +36.46% (p < 0.0001). This effect involved the SF-1 protein reduction: Pg: -36.34 ± 9.26%; tamoxifen: -46.25 ± 15.68% (p < 0.01). Finally, in a cohort of 36 ACC samples, immunohistochemistry showed undetectable/low level of ERs, while PgR demonstrated a higher expression. In conclusion, ACC experimental cell models expressed PgR and low levels of ER in line with data obtained in patient tissues, thus limiting the possibility of a clinical approach targeting ER. Interestingly, Pg exerted cytotoxicity also in metastatic ACC cells, although with low potency. Keywords: ACC cell lines; ACC primary cells; adrenocortical carcinoma; estrogen receptors; progesterone receptors; tamoxifen

1,25(OH)2 vitamin D(3) contributes to osteoclast-like trans-differentiation of malignant plasma cells

1,25-dihydroxyvitamin D (1,25(OH)2D) exerts pleiotropic effects including bone turnover and immune system regulation. It inhibits both T and B cell proliferation while decreasing the secretion of inflammatory cytokines and immunoglobulins. 1,25(OH)2D also modulates monocyte-macrophage and osteoclast (OC) maturation. Since we have previously described that malignant plasma cells may trans-differentiate towards the myeloid lineage participating to skeletal devastation in multiple myeloma (MM), we here evaluated in vitro the role of 1,25(OH)2D in this lineage switch. We investigated the gene and protein expression of vitamin D receptor (VDR) in MM cell lines. Thus, after cell treatment with 1,25(OH)2D, we analyzed their morphology and the expression of myeloid and OC markers. Finally, we assessed their bone resorption property on calcium phosphate slices. All MM cells expressed VDR in nuclear and perinuclear sites. Treatment with 1,25(OH)2D altered their morphology from round to fusiform, while inducing paxillin focalization. 1,25(OH)2D administration also up-regulated myeloid and OC genes, including C/EBPα RANK, M-CSFR and V-ATPase, whose promoters contain potential 1,25(OH)2D responsive elements. Finally, 1,25(OH)2D increased MM cell capability to generate pits of erosion on calcium phosphate discs. This data suggest that myeloma cells may undergo a functional trans-differentiation into OCs and, under appropriate experimental conditions, 1,25(OH)2D triggers this lineage switch

Cisplatin Cytotoxicity in Human Testicular Germ Cell Tumor Cell Lines Is Enhanced by the CDK4/6 Inhibitor Palbociclib

Background: Cisplatin-based chemotherapy is the mainstay of pharmacological treatment of testicular germ cell tumors (TGCTs) that, together with early diagnosis, surgery, and/or radiotherapy, has dramatically improved the prognosis. However, under the pressure of such pharmacological therapy (both classical cytotoxic drugs and targeted therapy), cancer cells may develop resistance. Thus, combination therapy that may include cytotoxic drugs and targeted therapy could offer an advantage to curing cancers. Here, we investigated the in vitro and in vivo antitumor activity of cisplatin, as a single-agent or in combination with palbociclib. Patients and methods: The cell viability of Ntera-2/cl.D1 (NT2/D1) and 833K after exposure to palbociclib and/or cisplatin was evaluated by MTT dye reduction assay and by ATPLite Luminescence Assay. Gene and protein expression was evaluated by quantitative reverse transcription polymerase chain reaction and by western blot. Flow cytometric cell-cycle analysis was performed, as well. The in vivo experiments were conducted on NT2/D1 xenografts in AB zebrafish embryos exposed to the drugs. Results: Palbociclib and cisplatin decreased TGCT cell viability both in vitro and in vivo. This effect was additive when cells were exposed to the drug combination. In the NT2/D1 cell lines, the drug combination also exerted a positive effect with regard to delaying cell recovery after the toxic insult. In the combination experiments, cisplatin-induced cell accumulation in G2/M was predominant compared with the palbociclib effect. Conclusions: These results could provide the rationale for developing further studies to improve the pharmacological treatment of TGCTs, but they must be demonstrated in a dedicated clinical trial

Cytotoxic Effect of Trabectedin In Human Adrenocortical Carcinoma Cell Lines and Primary Cells

Mitotane is the only drug approved for the treatment of adrenocortical carcinoma (ACC). The regimen to be added to mitotane is a chemotherapy including etoposide, doxorubicin, and cisplatin. This pharmacological approach, however, has a limited efficacy and significant toxicity. Evidence indicates that ACC seems to be sensitive to alkylating agents. Trabectedin is an anti-tumor drug that acts as an alkylating agent with a complex mechanism of action. Here, we investigated whether trabectedin could exert a cytotoxic activity in in vitro cell models of ACC. Cell viability was evaluated by MTT assay on ACC cell lines and primary cell cultures. The gene expression was evaluated by q-RT-PCR, while protein expression and localization were studied by Western blot and immunocytochemistry. Combination experiments were performed to evaluate their interaction on ACC cell line viability. Trabectedin demonstrated high cytotoxicity at sub-nanomolar concentrations in ACC cell lines and patient-derived primary cell cultures. The drug was able to reduce /β catenin nuclear localization, although it is unclear whether this effect is involved in the observed cytotoxicity. Trabectedin/mitotane combination exerted a synergic cytotoxic effect in NCI-H295R cells. Trabectedin has antineoplastic activity in ACC cells. The synergistic cytotoxic activity of trabectedin with mitotane provides the rationale for testing this combination in a clinical study

Neuroprotection by Cocktails of Dietary Antioxidants under Conditions of Nerve Growth Factor Deprivation

Dietary antioxidants may be useful in counteracting the chronic inflammatory status in neurodegenerative diseases by reducing oxidative stress due to accumulation of reactive oxygen species (ROS). In this study, we newly described the efficacy of a number of dietary antioxidants (polyphenols, carotenoids, thiolic compounds, and oligoelements) on viability of neuronal PC12 cells following Nerve Growth Factor (NGF) deprivation, a model of age-related decrease of neurotrophic support that triggers neuronal loss. Neuroprotection by antioxidants during NGF deprivation for 24 h was largely dependent on their concentrations: all dietary antioxidants were able to efficiently support cell viability by reducing ROS levels and restoring mitochondrial function, while preserving the neuronal morphology. Moreover, ROS reduction and neuroprotection during NGF withdrawal were also achieved with defined cocktails of 3–6 different antioxidants at concentrations 5–60 times lower than those used in single treatments, suggesting that their antioxidant activity was preserved also at very low concentrations. Overall, these data indicate the beneficial effects of antioxidants against oxidative stress induced by decreased NGF availability and suggest that defined cocktails of dietary factors at low concentrations might be a suitable strategy to reduce oxidative damage in neurodegenerative diseases, while limiting possible side effects

Palbociclib inhibits proliferation of human adrenocortical tumor cells

Adrenocortical cancer (ACC) is a rare malignant tumor. Pharmacological therapy is based on mitotane administered alone or in association with the EDP regimen (etoposide, doxorubicin, and cisplatin). However, the prognosis of ACC patients not amenable to surgery still remains poor and newer treatment strategies are needed. Drugs targeting cell cycle could be a relevant new therapeutic approach for patients with advanced ACC. Cell cycle is controlled by several key proteins, including CDKs (cyclin-dependent kinases), which are the target of recently discovered cell-cycle checkpoint inhibitors. Palbociclib is a CDK4/6-inhibitor that is active against a broad range of tumors and has an acceptable toxicity, being neutropenia being the most relevant side effect. This drug is actually approved in the management of locally advanced or metastatic breast cancer. In this study, we investigated in vitro the effect of Palbociclib in NCI-H295R ACC cells and human ACC primary cultures

Adrenocortical carcinoma xenograft in zebrafish embryos as model to study the in vivo cytotoxicity of abiraterone acetate

Abiraterone acetate (AbiAc) inhibits tumor growth when administered to immunodeficient mice engrafted with in vitro cell model of human adrenocortical carcinoma (ACC). Here, we developed and validated a zebrafish model engrafted with cortisol-secreting ACC cells to study the effects of AbiAc on tumor growth