Martina Fragni

GPNMB/OA protein increases the invasiveness of human metastatic prostate cancer cell lines DU145 and PC3 through MMP-2 and MMP-9 activity

Non-metastatic glycoprotein melanoma protein B (GPNMB), also known as osteoactivin (OA) is expressed in a wide array of tumors and represents an emerging target for drug development. In this study, we investigated the role of GPNMB/OA in the progression of human metastatic DU145 and PC3 prostate cancer cells. GPNMB/OA contribution in PCa malignant phenotype has been analyzed by small interfering RNA-induced GPNMB/OA silencing. We found that following GPNMB/OA silencing the migration capability of both DU145 and PC3 cells, evaluated by using in vitro invasivity assay, as well as the metalloproteinases MMP-2 and MMP-9 activity were equally strongly inhibited. By contrast knocking down GPNMB/OA weakly attenuated cell proliferation rate of DU145, an effect that paralleled with an increase number of apoptotic cells. However, PC3 cell growth seems to be not affected by GPNMB/OA. Together, these data reveal that GPNMB/OA acts as a critical molecular mediator promoting the acquisition of the more aggressive, pro-metastatic phenotype distinctive of human DU145 and PC3 cell lines.

The miR-21/PTEN/Akt signaling pathway is involved in the anti-tumoral effects of zoledronic acid in human breast cancer cell lines

Preclinical data indicate a direct anti-tumor effect of zoledronic acid (ZA) outside the skeleton, but its molecular mechanism is still not completely clarified. The aim of this study was to investigate the anti-cancer effects of ZA in human breast cancer cell lines, suggesting that they may in part be mediated via the miR-21/PTEN/Akt signaling pathway. The effect of ZA on cell viability was measured by MTT assay, and cell death induction was analyzed using either a double AO/EtBr staining and M30 ELISA assay. A Proteome Profiler Human Apoptosis Array was executed to evaluate the molecular basis of ZA-induced apoptosis. Cell cycle analysis was executed by flow cytometry. The effect of ZA on miR-21 expression was quantified by qRT-PCR, and the amount of PTEN protein and its targets were analyzed by Western blot. ZA inhibited cell growth in a concentration- and time-dependent manner, through the activation of cell death pathways and arrest of cell cycle progression. ZA downregulated the expression of miR-21, resulting in dephosphorilation of Akt and Bad and in a significant increase of p21 and p27 proteins expression. These results were observed also in MDA-MB-231 cells, commonly used as an experimental model of bone metastasis of breast cancer. This study revealed, for the first time, an involvement of the miR-21/PTEN/Akt signaling pathway in the mechanism of ZA anti-cancer actions in breast cancer cells. We would like to underline that this pathway is present both in the hormone responsive BC cell line (MCF-7) as well as in a triple negative cell line (MDA-MB-231). Taken together these results reinforce the use of ZA in clinical practice, suggesting the role of miR-21 as a possible mediator of its therapeutic efficacy.

Amiloride Is Effective in the Management of Abiraterone-Induced Mineralocorticoid Excess Syndrome without Interfering with Its Antineoplastic Activity

Background: The administration of abiraterone acetate (abiraterone) leads to an adrenocorticotropic hormone (ACTH)-driven increase in mineralocorticoid hormones, requiring glucocorticoid supplementation that may stimulate the growth of prostate cancer (PCa). Amiloride is a drug that selectively reduces the aldosterone-sensitive Na+/K+ exchange and could be effective in the management of mineralocorticoid excess syndrome (MCES). Methods: The efficacy of amiloride + hydrochlorothiazide (HCT) in the clinical management of abiraterone-induced MCES was assessed in 5 consecutive patients with castration-resistant PCa (CRPC). Then, using the in vitro experimental model of PCa cell lines, the possible effects of drugs usually used in the clinical management of CRPC patients on PCa cell viability were investigated. Results: Amiloride/HCT led to a complete disappearance of all clinical and biochemical signs of abiraterone-induced MCES in the 5 treated patients. The in vitro study showed that abiraterone treatment significantly decreased cell viability of both androgen receptor (AR)-expressing VCaP (vertebral-cancer of the prostate) and LNCaP (lymph node carcinoma of the prostate) cells, with no effect on AR-negative PC-3 cells. Prednisolone, spironolactone, and eplerenone increased LNCaP cell viability, while amiloride reduced it. The non-steroid aldosterone antagonist PF-03882845 did not modify PCa cell viability. Conclusions: The combination of amiloride/HCT was effective in the management of abiraterone-induced MCES. Amiloride did not negatively interfere with the abiraterone inhibition of PCa cell viability in vitro.

Palbociclib inhibits proliferation of human adrenocortical tumor cells

Adrenocortical cancer (ACC) is a rare malignant tumor [1]. Pharmacological therapy is based on mitotane administered alone or in association with the EDP regimen (etoposide, doxorubicin, and cisplatin) [1]. However, the prognosis of ACC patients not amenable to surgery still remains poor and newer treatment strategies are needed [2]. Drugs targeting cell cycle could be a relevant new therapeutic approach for patients with advanced ACC [3]. Cell cycle is controlled by several key proteins, including CDKs (cyclin-dependent kinases), which are the target of recently discovered cell-cycle checkpoint inhibitors [4]. 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 [5]. This drug is actually approved in the management of locally advanced or metastatic breast cancer [6]. In this study, we investigated in vitro the effect of Palbociclib in NCI-H295R ACC cells and human ACC primary cultures.

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

ABSTRACT 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/EBP&agr;, 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. HIGHLIGHTSMyeloma cells, under appropriate conditions, display an osteoclast‐like phenotype.In vitro, 1,25(OH)2‐D induces cytoskeleton remodeling in malignant plasma cells.1,25(OH)2‐D up‐regulates typical myeloid and osteoclast markers in MM cells.1,25(OH)2‐D reinforces the bone erosive activity of multiple myeloma cells.

Gpnmb/osteoactivin as a malignancy biomarker in an experimental model of human prostate adenocarcinoma

Background The goal is to analyze the role of Osteactivin (OA) in human prostate adenocarcinoma (CaP) cell lines, DU145 and PC3. OA plays a role in proliferation, adhesion, differentiation and protein synthesis in normal and malignant cells. Further, the OA expression is specific of activated mature osteoblasts. Methods DU145 and PC3 were maintained under recommended conditions and treated, respectively, with 50 ng/mL for 8 days and with 100 ng/mL for 15 days of Nerve Growth Factor (NGF). The NGF-induced reduction of invasive capacity was assessed by the BioCoat Matrigel Invasion Chambers technique. The gene expression was evaluated by Q-RT-PCR, while the protein expression by indirect immunofluorescence and by western blot. Results NGF treatment of DU145 and PC3 induced a reduction of 95% and 78% of the invasive capacity. NGF treatment decreased OA expression at both mRNA and protein levels. The OA was secreted into the culture medium. Conclusions DU145 and PC3 cells expressed OA, that is strongly reduced by NGF treatment. Since it is known that OA is involved in the acquisition of the cell invasive capability, it could thus be hypothesized that OA could be a factor that contributes to the acquisition of invasive properties of PCa and could be proposed as a biomarker of cell tendency to metastatization.

In vitro antitumor activity of progesterone in human adrenocortical carcinoma

PurposeThe management of patients with adrenocortical carcinoma (ACC) is challenging. As mitotane and chemotherapy show limited efficacy, there is an urgent need to develop therapeutic approaches. The aim of this study was to investigate the antitumor activity of progesterone and explore the molecular mechanisms underlying its cytotoxic effects in the NCI-H295R cell line and primary cell cultures derived from ACC patients.MethodsCell viability, cell cycle, and apoptosis were analyzed in untreated and progesterone-treated ACC cells. The ability of progesterone to affect the Wnt/β-catenin pathway in NCI-H295R cells was investigated by immunofluorescence. Progesterone and mitotane combination experiments were also performed to evaluate their interaction on NCI-H295R cell viability.ResultsWe demonstrated that progesterone exerted a concentration-dependent inhibition of ACC cell viability. Apoptosis was the main mechanism, as demonstrated by a significant increase of apoptosis and cleaved-Caspase-3 levels. Reduction of β-catenin nuclear translocation may contribute to the progesterone cytotoxic effect. The progesterone antineoplastic activity was synergically increased when mitotane was added to the cell culture medium.ConclusionsOur results show that progesterone has antineoplastic activity in ACC cells. The synergistic cytotoxic activity of progesterone with mitotane provides the rationale for testing this combination in a clinical study.