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Recent and growing literature has reported that oleuropein (OLE), the main polyphenol in olive leaf extract, inhibits tumor cell proliferation and reduces the invasiveness properties of cancer cells; therefore, OLE may play a significant role in the development of new drugs for cancer treatment. These antineoplastic properties have been reported in many experimental cancer models, but the effect of OLE on seminoma cells is yet to be evaluated. In the present study, we demonstrate, for the first time, that OLE reduces cell viability in both intra- and extragonadal TCAM-2 and SEM-1 seminoma cells, respectively, in a dose-dependent manner. As shown by Western-blot analysis, OLE exposure reduced cyclin-D1 expression and upregulated p21 Cip/WAF1 , concomitantly affecting the upstream pathway of NF-κB, leading to the reduction of its nuclear content, thereby suggesting that OLE could modulate cell-cycle regulators by inhibiting NF-κB. Moreover, Annexin V staining revealed that OLE induced apoptosis in cancer cells and upregulated the pro-apoptotic factor BAX. Through wound-healing scratch and transmigration assays, we also demonstrated that OLE significantly reduced the migration and motility of TCAM-2 and SEM-1 cells, and downregulated the expression of TGFβ-1, which is known to be the main pro-fibrotic factor involved in the acquisition of the migratory and invasive properties of cancer cells. Collectively, our results indicate that OLE reduces seminoma cell proliferation, promotes apoptosis, and counteracts cell migration and motility. Further studies are needed to explore the molecular mechanisms underlying these observed effects.

Although in past decades the adipokine leptin and its own receptor have been considered as significant cancer biomarkers, their potential involvement in human testicular seminoma growth and progression remains unexplored. Here, we showed that the expression of leptin and its receptor was significantly higher in human testicular seminoma compared with normal adult testis. Human seminoma cell line TCam-2 also expressed leptin along with the long and short isoforms of leptin receptor, and in response to leptin treatment showed enhanced activation of its downstream effectors. In line with these results, leptin stimulation significantly increased the proliferation and migration of TCam-2 cells. Treatment of TCam-2 cells with the peptide Leu-Asp-Phe-Ile (LDFI), a full leptin-receptor antagonist, completely reversed the leptin-mediated effects on cell growth and motility as well as reduced the expression of several leptin-induced target genes. More importantly, the in vivo xenograft experiments showed that LDFI treatment markedly decreased seminoma tumor growth. Interestingly, LDFI-treated tumors showed reduced levels of the proliferation marker Ki-67 as well as decreased expression of leptin-regulated genes. Taken together, these data identify, for the first time, leptin as a key factor able to affect testicular seminoma behavior, highlighting leptin receptor as a potential target for novel potential treatments in this type of cancer., (Copyright © 2019 American Society for Investigative Pathology. Published by Elsevier Inc. All rights reserved.)

Leydig cell tumors are the most frequent interstitial neoplasms of the testis with increased incidence in recent years. They are hormonally active and are considered one of the steroid-secreting tumors. Although usually benign, the malignant phenotype responds poorly to conventional chemotherapy or radiation, highlighting the need to identify new therapeutic targets for treatment. Here, we identified a novel glucocorticoid-mediated mechanism that controls cell growth in Leydig cell tumors. We found that a synthetic glucocorticoid receptor agonist, dexamethasone, reduces cell proliferation in rat Leydig tumor cells by decreasing the expression and the enzymatic activity of the estrogen-producing enzyme aromatase. This inhibitory effect relies on the ability of activated glucocorticoid receptor to regulate the aromatase gene transcriptional activity through the recruitment of nuclear receptor corepressor protein and silencing mediator of retinoid and thyroid hormone receptors to a newly identified putative glucocorticoid responsive element within the aromatase promoter II. Our in vivo studies reveal a reduction of tumor growth, after dexamethasone treatment, in animal xenografts. Tumors from dexamethasone-treated mice exhibit a decrease in the expression of the proliferation marker Ki-67 and the aromatase enzyme. Our data demonstrate that activated glucocorticoid receptor, decreasing aromatase expression, induces Leydig tumor regression both in vitro and in vivo, suggesting that glucocorticoid receptor might be a potential target for the therapy of Leydig cell tumors., (Copyright © 2016. Published by Elsevier Inc.)

Leydig cell tumors (LCTs) of the testis are steroid-secreting tumors associated with various steroid biosynthetic abnormalities and endocrine dysfunctions. Despite their overall rarity, LCTs are still of substantial interest owing to the paucity of information regarding their exact nature and malignant potential. In the present study, we disclose the ability of androgens to inhibit Leydig tumor cell proliferation by opposing to self-sufficient in situ estrogen production. In rat Leydig tumor cells, R2C, androgen treatment significantly decreases the expression and the enzymatic activity of cytocrome P450 aromatase, responsible for the local conversion of androgens into estrogens. This inhibitory effect relies on androgen receptor (AR) activation and involves negative regulation of the CYP19 gene transcriptional activity through the nuclear orphan receptor DAX-1 (dosage-sensitive sex reversal, adrenal hypoplasia critical region, on chromosome X, gene 1). Ligand-activated AR up-regulates the expression of DAX-1 and promotes its increased recruitment within the steroidogenic factor-1 site-containing region of the aromatase proximal promoter II in association with the nuclear receptor corepressor. The biological relevance in LCTs of the newly highlighted functional interplay between AR, DAX-1, and aromatase is underlined by our in vivo observations, revealing a marked down-regulation of AR and DAX-1 expression and a strong increase in aromatase levels in testes tissues from old Fischer rats with spontaneously developed Leydig cell neoplasia, compared with normal testes tissues from younger animals. In elucidating a mechanism by which androgens modulate the growth of Leydig tumor cells, our finding support the hypothesis that maintaining the adequate balance between androgen and estrogens may represent the key for blocking estrogen-secreting Leydigioma development, opening new prospects for therapeutic intervention.

Background: Estrogen signaling is mediated by estrogen receptor beta isoforms in normal and neoplastic human testes. Recently, a G-protein-coupled-receptor (GPER) has been suggested as being involved in rapid responses to estrogens in different normal and tumor cells., Methods: This study investigated the GPER expression in paraffin-embedded samples from non neoplastic and neoplastic human testes (sex-cord stromal and germ cell tumors) by immunohistochemical and Western Blot analyses., Results: In control testes, a positive GPER immunoreactivity was detected in Leydig and in Sertoli cells while all germ cells were immunonegative. Furthermore, neoplastic cells of the Sertoli cell tumor, Leydig cell tumor, seminoma and embryonal carcinoma samples were all immunopositive. The immunoblots of testis extracts confirmed the results., Conclusions: These findings suggest that GPER could mediate estrogen signaling in both normal and transformed somatic cells of human testis, but they reveal a differential expression of the novel estrogen receptor in non neoplastic and neoplastic germ cells.

Background: Estrogens exert a role on germ cell physiology of normal human testis through the mediation of the estrogen receptor (ER) beta subtypes. Epidemiological studies evidenced an increased incidence of testicular germ cell cancer after elevated pre-natal estrogen exposure but the expression of estrogen receptors in these testicular neoplasms has not been well elucidated., Methods: Immunohistochemistry and Western blot analysis were used to investigate the expression of three distinct ER isoforms, ERalpha, ERbeta1, and ERbeta2 in paraffin-embedded tissues from seminomas and embryonal carcinomas, which are the most common testicular germ cell tumours., Results: Neoplastic cells of all specimens revealed a positive ERbeta1 and ERbeta2 immunoreactivity, while the ERalpha signal was undetectable. A similar pattern of estrogen receptor immunostaining was also observed in the malignant germ cells of intratubular germ cell neoplasia, adjacent to testicular cancers. Western blot analysis of tumour extracts revealed two immunoreactive bands, a 59 kDa band for ERbeta1 and a 53 kDa band for ERbeta2., Conclusion: A variable ERbeta expression was previously reported in testicular germ cell tumours and, particularly, an ERbeta down-regulation was evidenced in seminoma and embryonal carcinoma. Conversely, the current study has clearly identified ERbeta1 and ERbeta2 in the neoplastic cells of seminoma and embryonal carcinoma, as well as in the malignant cells of their common pre-invasive precursor, intratubular germ cell neoplasia. Therefore, our findings suggest that ERbeta1, together with a possible ERbeta2 contribute, can mediate estrogen action in both early and late neoplastic testicular germ cells, not confirming the previously hypothesized antiproliferative effect of ERbeta on male gonadal cells.

A Leydig cell tumor is a rare neoplasm, deriving from the interstitial cells, whose pathogenesis has not been still defined. Leydig cells of normal adult testis are known as physiological targets for estrogens. However, some studies on transgenic rodents suggest a role of estrogens in the development of Leydig cell hyperplasia and Leydig cell tumor. Therefore, with the aim to evaluate a possible link between estrogens and testicular tumorigenesis, this study investigated the expression of aromatase and estrogen receptors (ERalpha, ERbeta(1), ERbeta(2)) in testes from two patients with Leydig cell tumor. A strong immunoreactivity for aromatase, ERbeta(1), and ERbeta(2), together with a detectable ERalpha immunostaining, was revealed in tumoral tissues. These findings were confirmed by western blot analysis of tumor extracts detecting a 55 kDa P450arom, a 67 kDa ERalpha band, a 59 kDa ERbeta(1) band, and a 53 kDa ERbeta(2) band. The pattern of ER expression in neoplastic cells appears different from that of control Leydig cells exhibiting only ERbeta(1) and ERbeta(2) isoforms. The authors hypothesize how the high estrogen production could play a role in the neoplastic transformation of Leydig cells, while the exclusive presence of ERalpha in tumoral cells could amplify estradiol-17beta signaling contributing to the tumor cell growth and progression.

Background: The enzyme cytochrome P450 aromatase, catalysing the conversion of androgens into estrogens, has been detected in normal human testicular cells suggesting a physiological role of local estrogen biosynthesis on spermatogenesis control. Estrogens, regulating cell growth and apoptosis, can also be involved in tumorigenesis process, but the possible link between estrogens and testicular neoplastic process is, up to now, scarcely known. This study examined aromatase expression in human seminoma, which is the most common germ cell tumour of the testis., Methods: The tumour-bearing testes were obtained from 20 patients with classic seminoma undergoing to therapeutic orchidectomy. Paraffin embedded tissues were processed for immunohistochemistry using a mouse monoclonal antibody generated against human placental cytochrome P450 arom, as primary antibody, and a biotinylated goat-anti-mouse IgG, as secondary antibody. Furthermore, Western blot analysis of seminoma extracts was carried out., Results: Intense P450 arom immunoreactivity was observed in the seminoma cells and Western blot analysis confirmed the immunodetection. A strong immunostaining was also detected in cells of intratubular germ cell neoplasia (IGCN), adjacent to seminoma., Conclusion: The present study demonstrated, for the first time in human, aromatase expression in neoplastic cells of seminoma suggesting a relation between local estrogen biosynthesis and germ cell tumorigenesis. The P450 arom immunolocalization in the cells of IGCN, representing the common precursor of most germ cell tumors, seems to support these findings.