Elsevier

European Journal of Cancer

Volume 40, Issue 14, September 2004, Pages 2064-2070
European Journal of Cancer

The distribution of drug-efflux pumps, P-gp, BCRP, MRP1 and MRP2, in the normal blood–testis barrier and in primary testicular tumours

https://doi.org/10.1016/j.ejca.2004.05.010Get rights and content

Abstract

The drug-efflux pumps P-glycoprotein (P-gp) and multidrug resistance-associated protein 1 (MRP1) are present in the blood–testis barrier (BTB) and may hamper the delivery of cytotoxic drugs to the testis. The precise localisation of P-gp and MRP1 in testicular tissue and the presence of the efflux pumps MRP2 and breast cancer resistance protein (BCRP) in the BTB are unknown. We therefore studied the localisation of these pumps in the BTB in normal testis (n=12), in non-seminoma (n=10) seminoma (n=10), and testicular lymphoma (n=9). Slides were scored semi-quantitatively for P-gp, MRP1, MRP2 and BCRP and blood vessels with factor VIII antibody. In normal testis, P-gp and BCRP were strongly expressed by myoid cells and luminal capillary endothelial wall and P-gp also by Leydig cells. MRP1 was observed at the basal side of Sertoli cells and on Leydig cells. MRP2 was only weakly expressed by myoid cells. Seminomas and non-seminomas expressed P-gp and/or BCRP and/or MRP1, lymphomas strongly expressed P-gp, weakly expressed BCRP and did not or showed weak expression of MRP1. There was very little staining for MRP2 in the tumours. Newly formed vessels in all tumours only expressed P-gp and BCRP. P-gp, BCRP and MRP1 are present in different cell layers of the normal testis, suggesting the optimal protection of spermatogenesis. In germ cell tumours, this expression pattern may explain the chemoresistance observed to P-gp, BCRP and MRP1 substrates. In germ cell tumours and testicular lymphomas, P-gp and BCRP expression by tumour cells and by newly formed vessels may also contribute to chemoresistance. These findings underscore the importance of removing the affected testis in cases of primary germ cell tumours and testicular lymphomas, irrespective of whether the patient has already undergone chemotherapy.

Introduction

The testes are a sanctuary for tumour cells during the administration of chemotherapeutic agents, such as vincristine and doxorubicin, for the treatment of lymphoblastic leukemias [1], [2]. Although complete remission to first-line chemotherapy is common in these patients, isolated testicular relapses and testicular combined with central nervous system (CNS) relapses have been reported [3]. Patient outcome following anthracycline-based chemotherapy for testicular lymphomas seems to be worse than for lymphomas at other sites, even after orchidectomy of the affected testis [4]. Metastatic germ cell tumours are extremely sensitive to chemotherapy regimens consisting of cisplatin, bleomycin and etoposide [5], but are, in general, resistant to cytotoxic agents that are known substrates of the drug-efflux pumps; P-glycoprotein (P-gp), breast cancer resistance protein (BCRP) or multidrug resistance protein 1 (MRP1). Cisplatin is a substrate of multidrug resistance protein 2 (MRP2) [6], [7]. However, after initial chemotherapy, in almost one third of patients, residual viable tumour remains at the primary tumour site [8], [9]. It is thought that the blood–testis barrier (BTB), which protects developing germ cells against harmful agents, hampers delivery of certain cytotoxic agents to the testis and tumours [10], [11]. The BTB consists of both a passive and active part with regard to drug transport. Three cell layers, endothelial cells, myoid cells and Sertoli cells, form the passive part of the BTB. The cells within these three layers are connected by tight junctions. The endothelial cells of the testicular capillaries lack fenestrations. Sertoli cells form the seminiferous tubule, in which germ cells divide and develop. The myoid cells form the Tunica propria, a thin layer around the seminiferous tubule [12], [13]. The active part of the BTB consists of efflux pumps, but the composition of this part is less well understood. It is already known that the efflux pumps, P-gp and MRP1, are expressed in the testis, but the expression of other pumps, such as MRP2 and BCRP, is unknown [10], [14], [15]. These members of the ABC transporter family are transmembrane proteins that are able to transport many substrates in an adenosine triphosphate (ATP) dependent manner. They transport a wide range of agents, such as cytotoxic drugs (vinca-alkaloids, anthracyclines, taxoids and epipodophyllotoxins), protease inhibitors, calcium antagonists and corticosteroids [16], [17]. Although there are differences in substrate specificity between P-gp, BCRP and MRP1 and 2, they have many substrates in common. MRP1 is expressed on the basolateral side of Sertoli cells [15], but the exact arrangement of these four efflux pumps in different cell layers of the BTB is unknown. Insight into their exact arrangement may help us to understand the function of the BTB in normal and malignant testicular tissues. For this reason, we elucidated in this study the localisation of the drug-efflux pumps, P-gp, BCRP, MRP1 and MRP2, in the different cell layers in normal testicular tissue and in previously untreated primary seminomas, non-seminomas and testicular lymphomas.

Section snippets

Chemicals

Biotinylated rabbit-anti-mouse secondary antibody (RAMBIO), rabbit-anti-rat (RARABIO), conjugated strepta-B-complex (ABC) and factor VIII antibody were purchased from DAKO (Glostrup, Denmark). The Avidin/Biotin blocking kit was from Vector Laboratories Inc. (Burlingame, CA, USA), 3.3-diaminobenzidine tetrahydrochloride from Sigma (St. Louis, MO, USA), phosphate-buffered saline (PBS: 6.4 mM Na2HPO4; 1.5 mM KH2PO4; 0.14 M NaCl; 2.7 mM KCl, pH 7.4) and imidazole from Merck (Darmstadt, Germany) and

Normal testicular tissue from normal testes

Representative photographs of the immunostaining results are presented in Fig. 1. The different cell types, which belong to the different layers of the BTB expressed in a reciprocal manner either BCRP and P-gp or MRP1. Table 1 presents the averaged semi-quantitative evaluation of P-gp, BCRP, MRP1 and MRP2 expression in the testicular tissue. P-gp and BCRP are expressed on the luminal side of the endothelial cells and on the apical side of the myoid cells. MRP1 is expressed on the basolateral

Discussion

In the present study, we elucidated the precise localisation of P-gp, BCRP, MRP1 and 2 in normal testes and in normal testicular tissues in the proximity of a primary testicular tumour. The expression of MRP2 in the BTB is negligible and will not be extensively discussed. The distribution of the other efflux pumps over different tissue layers suggests a histologically serial order of P-gp and BCRP versus MRP1. According to the detoxifying function of these pumps, it is suggested that the pump

Conflict of interest statement

All authors disclose any financial and personal relationships with other people or organisations that could inappropriately influence (bias) their work.

Acknowledgements

Supported by grant RUG 99-1882 of the Dutch Cancer Society and the Nijbakker Morra Foundation.

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