Transitional Cell Carcinoma Express Vitamin D Receptors

ORIGINAL ARTICLE ~

Transitional Cell Carcinoma Express Vitamin D Receptors Gregers G Hermann’ & Claus B Andersen2

‘Department of Urology, Herlev University Hospital, DK-2730 Copenhagen, Denmark. ‘Department of Pathology, Herlev University Hospital, DK-2730 Copenhagen, Denmark

(Submitted July 15, 1995. Accepted for publication June 11, 1996)

Scand J Urol Nephrol 31; 161-166, 1996

Recently, vitamin D analogues have shown antineoplastic effect in several diseases. Vitamin D analogues exert its effect by interacting with the vitamin D receptor (VDR). Studies of VDR in transitional cell carcinoma (TCC) have not been reported. The purpose of the present study was therefore to examine whether human bladder tumor cells express VDR. Tumor biopsies were obtained from 26 patients with TCC. Expression of VDR was examined by immunohistochemical experiments. All tumors expressed VDR. Biopsies from advanced disease contained more VDR positive cells than low stage disease (p < 0.05). Similarly, also tumor grade appeared to be related to the number of cells expressing the receptor. Normal urothlium also expressed VDR but only with low intensity. Our study shows that TCC cells possess the VDR receptor which may make them capable to respond to stimulation with vitamin D, but functional studies of vitamin D’s effect on TCC cells in vitro are necessary before the efficacy of treatment with vitamin D analogues in TCC can be evaluated in patients. Key words: bladder neoplasms, vitamin D, calcitriol receptors.

Gregers G Hermann, Department of Urology, Herlev University Hospital, DK-2730 Copenhagen, Denmark

During the past few years it has been known that vitamin D has important actions in addition to those associated with calcium metabolism. These actions which concern cellular differentiation, cell proliferation and modulation of the immune system (3, 19) may be used in cancer therapy.

Increased cell differentiation and antiproliferative effect of vitamin D have been observed in vitro in cell lines from lymphomas (16), rectal mucosa (37), colon cancer (33), endometrial cancer (1 l), prostate cancer (34) and breast cancer (9) and in vivo in rats (9). The anti-proliferative effect in breast and ovarian carcinoma cells has been associated to downregulation of the protooncogene c-myc (3 1). The immunological effects of vitamin D include a reduced proliferation, migration and IL-2 release of activated T-cells (5, 20, 21), increased differentiation of monocytes (30), changed production of tumor necrosis factor (TNF) and interferon (IFN) (24) and increased cytotoxicity .of lymphokine activated killer cells (29).

Vitamin D’s hypercalcaemic effect limits its therapeutic use. Vitamin D analogues with much less effect on the calcium metabolism than the native vitamin D have recently been developed. Some of these possess most of vitamin D’s remaining effects (4, 6, 25, 36).

One vitamin D analog has been used in humans to treat cutaneous metastatic breast cancer resulting in 4 partial responses in 16 patients after 6 weeks of topical treatment (7). A complete response of recurrent cutaneous T-cell lymphoma has also been reported after treatment with a topical vitamin D analog (32).

Vitamin D and its analogs mediate their effects by interaction with an intracellular vitamin D3 receptor (VDR) located both in the cytoplasm and the nucleus. The VDR belongs to the superfamily of transacting transcriptional factors, which includes the corticoster- oid and thyroid hormone receptors and the retinoid receptors (3, 19). VDR is present in many tissues including malignancies from lung, cervix, uteri, bone, skin, colon, lymphatic, ovarian, prostate and hema- poietic tissue (3, 23). It has not been reported whether transitional cell carcinoma (TCC) cells express VDR. Our study show that they do, but studies of the effect of vitamin D on TCC cells in vitro are needed before the therapeutic response to vitamin D analogues can be evaluated.

MATERIAL AND METHODS

Patients Biopsies were obtained from bladder tumors in 26

Scand J Urol Nephrol31 01996 Scandinavian University Press. ISSN 00365599

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162 G. G. Hermann & C. B. Andersen

I

Fig. I . Expression of vitamin D receptors in cells from the breast cancer cell line MCF7 used as positive control in the immunohistochemical experiments ( ~ 2 5 0 ) .

Fig. 2. Negative control. Grade III/IV tumor immunostained as described in text but without MAB1360 anti-VDR ( ~ 2 5 0 ) .

Scand J Urol Nephrol31

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Vitamin D receptors in TCC 163

Fig. 3. Papillary grade II/III transitional cell tumor expressing VDR (x250)

Ins

Fig. 4. Normal urothelium labelled with MAB1360 anti-VDR. The cells express VDR but less intensive than tumor cells ( ~ 2 5 0 ) .


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Table 1. Number of cells expressing vitamin D receptors ( W R + cells) in TCC related to tumor stage

Stage' pT314 Total No. ptt (%) No. ptt

PT2 No. ptt (%)

PT1 No. ptt (%)

VDR + PTa

0 0 0 0 0 0 1-33 7 (77) 2 (33) 1 (loo) 1 (10) 34-66 2 (23) 4 (67) 0 (0) 9 (90)

0 (0) 10 (loo) 0 (0) > 66 0 (0)

Total 9 (loo)

cells (%) No. ptt (%)

11 15 0

26 0 (0) l(lO0) 6 (100)

According to WHO.

patients (18 males, 8 females; mean age: 71.7 years; range: 48-85 years) with TCC and from normal urothelium in healthy subjects. The biopsies were immediately frozen to -80°C Celcius.

Immunohistochemical experiments. The expression of VDR was examined by immu-

nohistochemical experiments using the MAB1360 antibody (Cheminon International INC, Temecula, CA 92590, USA; antibody diluted approximately 1/ SO). MAJ31360 is a monoclonal rat IgG2b class antibody with high affinity and specificity for the VDR and which do not crossreact with other molecules (35). Peripheral blood mononuclear cells (PBMC) which do not express the VDR were obtained from one healthy subject and used as negative control in the experiments. All PBMC were purified from one blood sample by density-gradient centrifugation as previously described (13). After purification the PBMC were frozen (graduated freezing, 90% fetal calf serum, 10% Dimethylsulfoxide, liquid nitrogen). The breast cancer cell line MCF7 (9) was used as positive control (Fig. 1). The MCF7 cells were cultured in RPMI-1640 tissue culture medium supple- mented with 10% fetal calf serum. PBMC and MCF7 cells were cytocentrifuged to slides and after airdrying fixed in methanoVacetone (1/1) for 90 s. The biopsies were snap frozen to -80°C in liquid isopenthane. After sectioning into 4 microns thickness, tissue specimens were fixed in acetone for 10min. Immu- nostaining of tissue specimens, PBMC and MCF7 cells

were performed as previously described (1). Briefly, optimally diluted primary monoclonal MAB1360 anti- VDR was preincubated with human serum 5% in Tris buffer saline (Tl3S) and then applied to the tissue specimens and the cytocentrifuged cells for 30min followed by a two-step immunoperoxidase-technic using secondary and tertiary antibodies labeled with peroxidase. Three-amino-9-ethylcarbazole was used as substrate giving a bright red-brown coloured end- product. Background staining was minimal as shown in Fig. 2 which presents a grade III/IV tumor immunostained as described but without the monoclonal MAB1360 anti-VDR.

Evaluation of the staining was performed semi- quantitatively by estimating the number of positively reacting cells (VDR+ cells) as follows: 0: no VDR+ cells; +: 1-33% VDR+ cells; ++: 34-66% VDR' cells and + + +: >66% VDR+ cells.

STATISTICAL METHOD

The X2-test for more than 2 independent samples were used to evaluate difference in VDR expression between patients with different tumor stage and tumor grade. The significance level was 5%.

RESULTS

VDR+ cells were present in all tumors, but no tumors contained more than 66% VDR+ cells. A papillary

Table 11. Number of cells expressing vitamin D receptors (VDR + cells) in TCC related to tumor grade

Grade'

V D R + 1 2 3 4 Total cells (%) No. ptt (%) No. ptt (%) No. ptt (%) No. ptt (%) No. ptt

0 0 0 0 0 0 11 15

1-33 0 (0) 7 (77) 3 (30) 1 (17)

0 34-66 1 ( W 2 (33) 7 (70) 5 (83)

26 0 (0) > 66 0 (0) 0 (0)

Total l(lO0) 9 (100) 6 (100) 0 (0)

10 (100)

' According to Bergquist et al. (2).


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Vitamin D receptors in TCC 165

grade II/III tumor labelled with MAE31360 anti-VDR is presented in Fig. 3. The VDR was identified both in the cytoplasm and the nucleus. Table I shows that more cells express VDR in advanced disease than in low stage disease (p < 0.05). The data also indicate that tumor grade is related to the number of VDR+ cells (Table 11) but this relation was not statistically significant.

Figure 4 shows that normal urothelium also express the VDR but less intensive than the tumors.

DISCUSSION

This study shows that normal urothelium and TCC cells express VDR, an observation which has not been reported previously. Our data furthermore show that the number of cells expressing VDR increases with tumor stage and possibly with tumor grade. This is in accordance with the observation that normal urothelium express less VDR than transitional cell tumors. These findings suggest that the increased expression of VDR in TCC is a result of the neoplastic activity of the tumor cells.

The use of treatment with vitamin D analogues may therefore be a possibility in TCC. The antiproliferative properties of vitamin D may reduce the growth of tumor cells in patients with TCC including patients with intraepithelial neoplasia without concurrent tumor. This effect may even be most pronounced in high-stage and high-grade disease as the number of VDR positive cells appear to be highest in these tumors.

TCC is associated with impaired immunological reactivity (8, 14, 18, 27) and several treatments stimulating the immune system have given substantial response in TCC (10, 15, 17, 22, 28). The function of monocytes appear to be essential in the immunological reaction against urological cancer (26). Vitamin D analogues increase the differentiation of monocytes and interfere with their production of cytokines (12) and may through this mechanism exert a therapeutic effect in TCC. However, further studies of vitamin D’s effect on TCC cells in vitro are necessary before the efficacy of vitamin D analogues in TCC can be evaluated in patients.

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Transitional Cell Carcinoma Express Vitamin D Receptors