Abstract
Testicular germ cell tumours (TGCTs) are the most common malignancy in young men aged 18-35 years. They are clinically and histologically subdivided into seminomas and non-seminomas. 1,25-Dihydroxyvitamin D (1,25(OH)2D3) is the active form of vitamin D and exerts its actions via a specific intracellular vitamin D receptor (VDR). Several investigations in the recent years have revealed, in addition to a physiological occurrence of the VDR in various tissues, VDR expression in different human malignancies. Furthermore, 1,25(OH)2D3 plays an important role in the regulation of cell proliferation and differentiation. In different normal and malignant cell types, antiproliferative and pro-differentiating effects of 1,25(OH)2D3 are described. We investigated whether TGCT express the VDR, wether differences exist between the histological subtypes and if vitamin D has a function on the proliferation of tumour cells. Furthermore, we investigated the potential function of the vitamin D-regulated genes nuclear receptor co-repressor 1(NCOR1), nuclear receptor co-repressor 2 (NCOR2), thyroid receptor interacting protein 15 (TRIP15), Growth Arrest and DNA Damage (GADD45), MAP kinase-activated protein kinase 2 (MAPKAPK2), Cytochrome P450, family 24, subfamily A, polypeptide 1 (CYP24A1) and Cytochrome P450, family 27, subfamily B, polypeptide 1 (CYP27B1) in the pathogenesis of TGCT. We demonstrate, for the first time, that primary TGCT as well as TGCT cell lines, express VDR mRNA and protein. Vitamin D and VDR may play a role in the pathogenesis of TGCTs. Furthermore, vitamin D inhibits proliferation of TGCT cell-lines, potentially via an increase in expression of GADD45. Our data suggest that vitamin D could play a role in antitumour therapy.
Testicular germ cell tumours (TGCTs) represent the most common malignant tumours in men aged 18 to 35 years and comprise 98% of all testicular malignancies. The incidence is constantly increasing in Europe as well as in the U.S.A., having doubled over the last 40 years (1). Histologically, TGCTs are divided into two main groups, seminomas and nonseminomas, the nonseminomas being subdivided into embryonal carcinomas, yolk sac tumours, chorioniccarcinomas and teratomas (2). All invasive testicular germ cell tumours arise from intratubular germ cell neoplasia, unclassified type, (IGCNU) which represent the precursor lesions of invasive TGCT (3).
1,25-Dihydroxyvitamin D (1,25(OH)2D3) is the active form of vitamin D and exerts its action via a specific intracellular vitamin D receptor (VDR). The VDR belongs to the steroid, thyroid and retinoid receptor gene family and regulates the transcriptional activity of 1,25(OH)2D3-influenced genes by complexing with vitamin D-responsive elements (VDREs). VDREs are located in the promoter region of DNA of target genes and are able to induce the transcription of the target genes (4-6). Several investigations have shown VDR expression in different tumour types, including leukemia cells, colon and breast cancer cells, prostate carcinoma cells and thyroid carcinomas (7-11). Besides the action of 1,25(OH)2D3 on calcium and phosphate homeostasis, it has been demonstrated that 1,25(OH)2D3 is a significant modulator of cellular differentiation and proliferation in a number of different normal and malignant cells (12).
In regulating the cell cycle, signal transduction and interaction with thyroid hormone receptor in addition to the VDR, different genes are involved. Cytochrome P450, family 27, subfamily B, polypeptide 1 (CYP27B1) and Cytochrome P450, family 24, subfamily A, polypeptide 1 (CYP24A1) are two key enzymes of 1,25(OH)2D3 synthesis and act at different points in regulating the metabolism of vitamin D3 (6). Nuclear receptor co-repressor 1(NCOR1) and nuclear receptor co-repressor 2 (NCOR2) are nuclear co-repressors which support nuclear receptors in the down-regulation of target gene expression (13, 14). The protein thyroid receptor interacting protein 15 (TRIP15), also called CSN2, is involved as part of COP9/signalosome complex in the regulation of different cell-signalling pathways and the cell cycle. It influences ligand binding to thyroid hormone and retinoic acid X receptors (RXR) (15, 16). MAP kinase-activated protein kinase 2 (MAPKAPK2) is involved in p38 MAPK signalling pathway and regulates gene expression (18, 19). Growth Arrest and DNA Damage (GADD45) is involved in the replication of DNA, the G1 cell cycle arrest and repression of proto-oncogenes in exerting antiproliferative effects in tumour cells. It directly affects proapoptotic actions and functions of DNA repair (20, 21). We investigated these genes for their potential role in the pathogenesis of TGCT. Furthermore, we analysed the expression and function of VDR and its co-regulators in primary TGCT and two TGCT cell lines.
Materials and Methods
Tissue samples of primary TGCT. Tumour tissues were acquired from 80 patients from the Federal Armed Forces Hospital, Hamburg, Germany. The mean age of patients was 33.86 years. An overview of patient ages and the different tumour types is presented in Table I. Ethical approval for using the human material in the present study was obtained from the Ethics Committee of the Federal Armed Forces Hospital, Hamburg. Tumour tissues from each testis were immediately fixed in HOPE (Hepes–glutamic acid buffer-mediated organic solvent protection effect) solution (DCS Diagnostic Systems, Hamburg, Germany) as described previously (22, 23).
Immunohistochemistry. Paraffin sections were used to perform the immunohistochemical reactions for VDR. The sections used were dewaxed, rehydrated and incubated with a polyclonal antibody against human VDR for one hour, at room temperature, in 1:200 dilution (Santa Cruz, CA, USA). Afterwards, the sections were incubated with the REAL Link/Label detection system (Dako, Hamburg, Germany) as described by the manufacturer. The signals were visualised with 3,3-diaminobenzidine (DAB; Dako). All sections were analysed by light microscopy after counterstaining with Meyer's hematoxylin. The immunohistochemical staining was evaluated according to the Remmele score in the diagnosis of breast cancer using immunohistochemical multiplying scores. This is the product of the percentage of positive tumour cells and the staining intensity (Table II) (24).
RNA isolation and quantitative real time polymerase chain reaction (qRT-PCR). RNA from HOPE-fixed paraffin-embedded tissues and TGCT cells were isolated with the RNeasy Mini Kit (Qiagen, Hilden, Germany). RNA integrity and quantity were measured with an Agilent Bioanalyzer 2100 using an RNA 6000 Nano LabChip-Kit (Agilent Technologies, Waldbronn, Germany). For reverse transcription, 500 ng of total cellular RNA were transcribed with random hexamer primers using an Omniscript RT Kit (Qiagen). Analysis of gene expression was performed with an iCycler iQ real-time detection system (Bio-Rad, Munich, Germany) and the QuantiTectSYBR Green Kit (Qiagen). The 20 μl reaction mixture from the kit was supplemented with 2 μl cDNA and 0.6 μM gene-specific primers. Primers (MWG, Ebersberg, Germany) were designed using the Primer 3 on-line primer design programme (http://frodo.wi.mit.edu/primer3/) and the Operon oligo tool kit (http://www.operon.com).The primer sequences used are shown in Table III.
Culture and drug treatment of TGCT cell lines. The human TGCT cell lines used in the present study were NTERA-2 (CRL 1973) and NCCIT (CRL 2073) (American Type Culture Collection, Manassas, VA, USA). The explored cell lines were maintained in HEPES-buffered RPMI-1640 (Biochrom, Berlin, Germany) supplemented with foetal calf serum (FCS, 10%; CC Pro, Neustadt,Germany),penicillin (100 IU/ml; Sigma, Munich, Germany),streptomycin (100 μg/ml; Sigma) and L-glutamine (2 mM; Biochrom, Berlin, Germany). The incubation temperature was 37°C in a humid atmosphere with 5% carbon dioxide in air. For stimulation with 1,25(OH)2D3, the cells were plated sub-confluently in 75 ml flasks and cultured in serum-free medium for one day prior to treatment, the latter step to synchronize the cells and exclude influence of serum components on proliferation of tumour cells.
Proliferation assay. The proliferation of the TGCT cell lines was measured after stimulation with 50,100 or 200 nM 1;25(OH)2D3 for 24 and 48 h with a BrdU-proliferation assay (Roche, Mannheim, Germany) as described by the manufacturer. The absorbance was determined in an ELISA reader (BioRad, Munich, Germany) at a wavelength of 450nm and a reference length from 655nm.
Western blotting. Tumour cells were harvested at 107 cells in 500 μl lysis buffer (0.4% sodium deoxycholate, 1% NP-40, 50 mM EGTA, pH 7.4, 10 mM Tris, pH 7.4, 1 mM phenylsulphonyl fluoride (PMSF), 10 μg/ml leupeptin, 10 μg/ml aprotinin). Thereafter, cell proteins were isolated as described previously (25).
Results
TGCTs express VDR. For expression analysis of the VDR protein, we investigated HOPE-fixed and paraffin-embedded TGCT tissue samples by immunohistochemistry. The tumour-free normal testicular tissue (n=80) showed no expression of VDR in the germ cells or cells of sex cord stroma (Leydig cells, Sertoli cells). IGCNU (n=37) as a non-invasive TGCT precursor lesion, exhibited slight cytoplasmatic expression of VDR in the atypical germ cells. In all examined seminomas (n=57), only slight cytoplasmatic expression of VDR protein was detected in the tumour cells (Figure 1A). All 23 examined nonseminomas, embryonic carcinoma (n=13), teratomas (n=8), chorionic carcinoma (n=1) and yolk sac tumours (n=1) exhibited strong cytoplasmatic and nuclear VDR expression (Figure 1B). Table IV gives an overview of the tissues divided into the different groups, investigated by immunohistochemistry. TGCTs were also analysed by qRT-PCR. An expression of VDR mRNA was demonstrated in TGCT but not in tumour-free testis. Seminomas exhibited a moderate expression of VDR, whereas nonseminomas exhibit a significantly higher expression of the VDR in the investigated tissues (data not shown).
VDR expression in TGCT cell lines. In addition to the studies in 80 primary TGCTs, the expression of VDR was investigated in the TGCT cell lines NCCIT and NTERA-2 by qRT-PCR. The qRT-PCR showed that the tumour cell lines express VDR mRNA (Figure 2A). To verify the results on translational level, VDR protein expression was examined in the TGCT cell lines NCCIT and NTERA-2 by western blot. Both TGCT cell lines express VDR protein (Figure 2B).
Proliferative behaviour of NCCIT and NTERA-2 cells after vitamin D stimulation. The proliferative behaviour of the two TGCT cell lines NCCIT and NTERA-2 was investigated after stimulation with 50, 100 or 200 nM of 1,25(OH)2D3 for 24 and 48 h. After 24 h, the tumour cell line NCCIT exhibited a significant decrease in proliferation by up to 44% (p<0.05) at a concentration of 200 nM 1,25(OH)2D3 and a reduction of proliferation by 13% after 48 h stimulation with 200 nM (Figure 3A). The proliferative behaviour after 24 h of the TGCT cell line NTERA-2 exhibited a statistically significant decrease up to 39% (p<0.05) at a concentration of 200 nM vitamin D and after 48 h stimulation with 200 nM 1,25(OH)2D3 proliferation was reduced up to 9% (Figure 3B).
Vitamin D3-regulated genes in TGCT cell lines. After stimulation with 1,25(OH)2D3, the tumour cell lines NCCIT and NTERA-2 were investigated by qRT-PCR for an expression of the vitamin D3-regulated genes VDR, NCOR1, NCOR2, TRIP15, GADD45, MAPKAPK2, CYP24A1 and CYP27B1. The stimulation was performed with 50, 100 or 200 nM 1,25(OH)2D3 for 24 h. After stimulation with 50 and 100 nM 1,25(OH)2D3, the tumour cell line NCCIT showed no statistical change in the expression of the examined genes. After stimulation with 200 nM 1,25(OH)2D3 VDR, NCOR1 and MAPKAPK2 gene expression significantly increased, whereas that of NCOR2 statistically decreased (Figure 4A). After stimulation with 50 and 100 nM 1,25(OH)2D3, the tumour cell line NTERA-2 sustained no statistical change in the expression of the examined genes. After stimulation with 200 nM 1,25(OH)2D3 of the NTERA-2 cell line, there was a further significant rise in expression of VDR and GADD45, while CYP27B1 gene expression decreased (Figure 4B).
Discussion
Expression of VDR in TGCT. In addition to the physiological occurrence of the VDR in various tissues, several research groups have demonstrated the presence of VDR in various malignancies in recent years. VDR expression has been studied in early and well-differentiated colon carcinoma (8), breast cancer (9), in prostate cancer (10), in papillary thyroid carcinoma (11), basal cell carcinoma (17), and in tumours of the lung (26). The results presented here show the expression of VDR in TGCT for the first time.
Early and well-differentiated colon carcinomas express VDR. Interestingly, this VDR expression was largely lost in late stages and poorly differentiated colon carcinomas (27, 28). This supports the hypothesis of Khadzkou et al. (11), that 1,25(OH)2D3 in connection with the VDR may have a promoting effect on the differentiation and proliferation of tumour cells. The variety of different tumour types in which VDR is expressed speaks for its importance in differentiation and proliferation in the pathogenesis of tumours.
Proliferative behaviour of TGCT cell lines in response to vitamin D stimulation and therapeutic aspects of vitamin D. In a variety of normal and malignant cell types, antiproliferative and pro-differentiating effects of 1,25(OH)2D3 have been shown (29). In studies of prostate and breast cancer, significant antitumour effects of 1,25(OH)2D3 and vitamin D analogues are reported (30, 31). 1,25(OH)2D3 and its derivatives have an effect on the regulation of proliferation, apoptosis and angiogenesis (32, 33). Here, we demonstrate a significant antiproliferative effect while stimulating the TGCT cell lines NCCIT and NTERA-2 with 1,25(OH)2D3. The described antiproliferative effects open up possible opportunities for a potential pharmacological use of 1,25(OH)2D3 for patients with TGCT.
The role of GADD45 in the decrease of proliferation after stimulation with 1,25(OH)2D3. The observations regarding the expression of GADD45 are also noteworthy. 1,25(OH)2D3 induces up-regulation of GADD45 in ovarian tumours, colon cancer and leukaemia cells. GADD45 is involved in the activation of various genes. These genes are involved, among other functions, in the replication of DNA and G1 cell cycle arrest, and by repression of proto-oncogenes, GADD45 has antiproliferative effects in tumor cells. It acts directly proapototically and affects DNA repair (34). Both investigated TGCT cell lines, NCCIT and NTERA-2, express GADD45. Interestingly, there is an up-regulation of GADD45 in both TGCT cell lines after stimulation with 1,25(OH)2D3. These findings may indicate that 1,25(OH)2D3 has the ability to increase the expression of GADD45 and its effect on differentiation and growth of TGCT. Interestingly, vitamin D directly induces an increase in transcription of GADD45. The increase in GADD45 protein expression leads to a decrease of cyclin B, the regulatory subunit of Cyclin-dependent kinase 1 (encoded by gene CDC2). This induces a cell cycle arrest in the transition from the G2 into the M phase (21, 35). This mechanism may also explain the decrease in proliferation after vitamin D stimulation in both investigated TGCT cell lines (Figure 5).
Regulation of vitamin D3 polymorphism in TGCT. As described above, NCOR1 and NCOR2 are nuclear co-repressors which support nuclear receptors in the down-regulation of target gene expression. Tzelepi et al. (36) correlated NCOR1 expression with morbidity-free interval. It seems that the impaired regulation of vitamin D3-induced genes such as NCOR1 contributes to the initiation and progression of colorectal cancer (36). The expression of NCOR1 in TGCT demonstrated here may play a role in a better prognosis for patients with TGCT, especially in nonseminoma. The decrease of NCOR2 gene expression in both TGCT cell lines after stimulation with 1,25(OH)2D3 could have a regulatory effect on the interaction between VDR/retinoic acid-X receptors (RXR).
TRIP15 is a protein involved in the regulation of various cell signalling pathways and the cell cycle. It also has an influence on thyroid hormone and RXR (37). Both investigated TGCT cell lines express TRIP15, but there was only a slight increase of mRNA expression in the NCCIT cell line after stimulation with 1,25(OH)2D3. Whether TRIP15, as a subunit of the CSN2 COP9/signalosome (38), influences the transcription of the VDR has not been studied so far. These findings, however, could be used as an approach for further investigations.
Kumar et al. (39) demonstrated the role of MAPKAPK2 in bladder cancer and confirmed the results presented by Xu and colleagues on the role of MAPKAPK2 in the invasion of prostate cancer (40). Kotlyarov and colleagues (41) also underlined the importance of MAPKAPK2 in the metastasis of tumour cells. Interestingly with regard to the TGCT cell lines expressing MAPKAPK2, there was a significant increase in MAPKAPK2 mRNA concentration after stimulation with 1,25(OH)2D3 in the NCCIT cell line. MAPKAPK2 expression in TGCT cell lines may play a role in the pathogenesis of TGCT and may have an effect in metastasis.
CYP27B1 and CYP24A1 are two key genes in the regulation of vitamin D3 metabolism. In our examination of NTERA-2, we recorded expression of CYP24A1 after stimulation with vitamin D3. However vitamin D3 had no effect on the protein concentration of CYP24A1 in the
TGCT cell lines in cell culture experiments. CYP24A1 is not expressed in the tumour cell line NCCIT. Besides its occurrence in the kidneys, 1α-hydroxylase is also found in extrarenal tissues. It is possible that local conversion of 25(OH)D3 to 1,25(OH)2D3 takes place in various tissues such as colon, brain, placenta, pancreas, lymph nodes and skin. In the early stages of well-differentiated colon cancer, there is an increased expression of CYP27B1. In poorly differentiated stages of colon cancer, a decrease of CYP271 expression was observed (28, 42). Increased expression of CYP27B1 in cancer tissue speaks for a local conversion of 25(OH)D3 to 1,25(OH)2D3 and, in theory, 25(OH)D3 and 1,25(OH)2D3 have a chemo preventative role in these tumours (43). An increased CYP24A1 mRNA expression in tumours results in reduced 1,25(OH)2D3 concentration. A high CYP24A1 mRNA expression may counteract the antiproliferative effect of 1,25(OH)2D3.
Cross et al. (44) showed that the increased expression of CYP24A1 would support a simultaneous reduction of the gene product of CYP27B1 in high-grade colon cancer. While only a low expression of CYP24A1 mRNA was recorded in normal ovary, lung, colon and breast tissues, increased expression of CYP24A1 mRNA occurred in tumour tissues from lung, ovary and colon. In tumour tissue of the breast, however, there was a decrease in expression. This suggests that 1,25(OH)2D3 levels might be reduced in these cases (44-48).
In conclusion, our data suggest that TGCT express VDR. While IGCNU and seminomas exhibited only a slight cytoplasmic expression of VDR, all investigated nonseminomas had a strong cytoplasmic and nuclear expression. The data also suggest that vitamin D could have an inhibitory effect on proliferation via an increase expression of GADD45 and therefore might have a role in antitumour therapy. We demonstrated that primary TGCT and TGCT cell lines express VDR and that the stimulation of TGCT cells with 1,25(OH)2D3 leads to up-regulation of VDR and inhibition of proliferation in tumour cells.
Acknowledgements
The Authors thank Anke Klages for technical assistance.
Footnotes
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Conflict of Interest Statement
In regard to the present publication, there is no conflict of interest with other institutions or companies.
- Received September 29, 2011.
- Revision received November 2, 2011.
- Accepted November 3, 2011.
- Copyright© 2012 International Institute of Anticancer Research (Dr. John G. Delinassios), All rights reserved