Cancer Therapy Vol 2, 345-352, 2004

The peripheral benzodiazepine receptor: A target for innovative diagnostic and therapeutic approaches in gastrointestinal oncology

Review Article

Kerstin Maaser, Andreas P. Sutter, and Hans Scherübl*

Medical Clinic I, Charité – Universitätsmedizin Berlin, Campus Benjamin Franklin, 12200 Berlin, Germany

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*Correspondence: Hans Scherübl, Medical Clinic I, Charité – Universitätsmedizin Berlin, Campus Benjamin Franklin, Hindenburgdamm 30, 12200 Berlin, Germany, phone: +493084453534, fax: +493084454481, e-mail: hans.scherubl@charite.de

Key words: gastrointestinal oncology, peripheral benzodiazepine receptor (PBR)

Abbreviations: diazepam binding inhibitor, (DBI); extracellular signal-regulated kinase, (ERK); growth arrest and DNA damage protein, (gadd); mitogen-activated protein kinase, (MAPKinase); peripheral benzodiazepine receptor, (PBR); permeability transition pore, (PTP); positron emission tomography, (PET)

Received: 27 August 2004; Accepted: 13 September 2004; electronically published: September 2004

Summary

The peripheral benzodiazepine receptor (PBR) plays a role in the regulation of cellular proliferation, immunomodulation, porphyrin transport and heme biosynthesis, regulation of steroidogenesis and apoptosis. The PBR has been implicated in the growth control of various cancers. The PBR displays antiapoptotic and proliferative functions. PBR was found to be overexpressed in several cancers including colorectal, breast, or brain cancers. In colorectal cancer, high PBR overexpression correlates with poor prognosis. PBR-specific ligands were shown to have antiproliferative effects in cancer cells. This review focuses on the functional role of PBR in gastrointestinal cancers. It describes the PBR expression in normal and neoplastic gastrointestinal tissues, presents recent reports on possible diagnostic and prognostic applications of PBR and its ligands, and discusses PBR- and PBR ligand-based therapeutic approaches.

I. Introduction

The peripheral benzodiazepine receptor (PBR) was first described as a high affinity binding site for diazepam in rat kidney (Braestrup and Squires, 1977). In fact, the receptor is ubiquitously expressed in peripheral tissues as well as in the brain but to very different extents. Its expression levels range from very high in steroid-producing tissues to relatively low in the brain, breast or gut mucosa. The PBR is an evolutionary conserved 18 kD protein which contains five transmembrane domains. It is mainly localized in the outer mitochondrial membrane (Anholt et al, 1986), but has also been detected in the plasma membrane (Garnier et al, 1993), and in the nucleus (Hardwick et al, 1999). In the mitochondrial membrane, PBR is associated with the voltage dependent anion channel (VDAC) and the adenine nucleotid translocator (ANT). VDAC and ANT are known to be part of the permeability transition pore (PTP) which is involved in the initiation and regulation of apoptosis.

PBR has been implicated in various cellular processes, including steroidogenesis, immune response, apoptosis, and proliferation (reviewed in Beurdeley-Thomas et al, 2000; Casellas et al, 2002; Papadopoulos, 2003).

Different endogenous compounds have been identified as ligands of the PBR. The diazepam binding inhibitor (DBI) is a polypeptide which binds to PBR with intermediate affinity (Bovolin et al, 1990). Other PBR binding molecules include the porphyrins protoporphyrin IX, mesoporphyrin, and hemin (Verma et al, 1987). Moreover, PBR binds cholesterol and is involved in its transport to the inner mitochondrial membrane which is the rate-limiting step of steroid biosynthesis (Papadopoulos et al, 1997). Specific synthetic ligands have been widely used to pharmacologically characterize the PBR: the benzodiazepine 4-chlorodiazepam (Ro5-4864), the isoquinoline carboxamide PK 11195, and the indoleacetamide FGIN-1-27, all of which bind with high affinity to the PBR but not to the central benzodiazepine binding site, the GABA_A receptor (Le Fur et al, 1983; Kozikowski et al, 1993).

The PBR has been implicated in growth control of various cancers for two reasons: First, PBR has been shown to be overexpressed in a variety of tumors, and second, specific PBR ligands inhibit cancer cell proliferation. Proliferative and antiapoptotic properties have been ascribed to PBR. Direct evidence for the antiapoptotic action of PBR was provided by the findings that transfection and subsequent overexpression of PBR protected cells against UV- or hydrogen peroxide-induced apoptosis (Carayon et al, 1996; Stoebner et al, 2001). Moreover, PBR knockout Leydig cells grew much slower than their wild-type counterparts (Amri et al, 1999). In breast cancer cell lines, the level of PBR expression correlated inversely with the doubling time (Beinlich et al, 2000) and positively with the ability of the tumors to grow in SCID mice (Hardwick et al, 2001).

Gastrointestinal cancers comprise biologically different entities. The aim of this report is to give an overview about the occurrence and functional role of PBR in colorectal adenocarcinoma, esophageal adenocarcinoma and squamous cell cancer, and hepatocellular carcinoma. Innovative PBR-based diagnostic, prognostic, and therapeutic approaches will be described.

II. PBR expression in gastrointestinal cancers

The association of PBR with cancer development was first suggested when the binding capacities for PBR-specific ligands were found to be increased in cancers in comparison to benign tissues. An increase in PBR binding was detected in a variety of cancers including cancers of the colon (Katz et al, 1990b), liver (Venturini et al, 1998), ovary (Katz et al, 1990a), brain (Cornu et al, 1992), and breast (Beinlich et al, 1999).

In the gastrointestinal tract, PBR expression is low in the mucosa of the esophagus (Sutter et al, 2002) and the colorectum (Maaser et al, 2002; Han et al, 2003; Bribes et al, 2004) (Figure 1). In the small intestine, PBR was found to be heterogeneously expressed and a strong expression was detected in the mucosa of the stomach (Bribes et al, 2004; Ostuni et al, 2004). In tissues with a low basal expression, PBR was found to be overexpressed upon neoplastic transformation.

Figure 1. Immunohistochemical detection of PBR overexpression. Immunohistochemical assessment of PBR expression in gastrointestinal cancers in comparison to normal tissues of the same patient. Sections of normal colorectal mucosa (A) and colorectal carcinoma (B), normal esophageal squamous mucosa (C) and esophageal squamous cell carcinoma (D), and liver tissue (E) with non-neoplastic components (no) and hepatocellular carcinoma (HCC) were incubated with monoclonal anti-PBR antibody 8D7. Specific antibody-binding was detected using the APAAP “fast-red system”. Bar = 20 mM (B, D), bar = 50 mM (E).

The frequency of PBR overexpression is tissue specific. In esophageal squamous cell carcinoma only one third of the patients showed an increase in PBR expression in cancer in comparison to the normal squamous epithelium (Sutter et al, 2002). In patients with colorectal carcinoma 88% of the cancers expressed more PBR than the normal colorectal mucosa of the same patient (Maaser et al, 2002). Preliminary data of our group suggest that PBR up-regulation is an early event in colorectal carcinogenesis. PBR overexpression was observed already in early adenomas and persisted even in metastases of colorectal cancers. The results concerning PBR expression in liver tissues are variable. Different groups detected no or weak PBR expression in normal liver tissue but increased in hepatocellular carcinoma (HCC) (Figure 1) (Venturini et al, 1998; Venturini et al, 1999; Sutter et al, 2004b; Bribes et al, 2004). In contrast, Han et al. (2003) detected a heterogenous, partly strong PBR expression in normal liver tissues and no further increase in hepatocellular carcinomas.

The increase of PBR expression in cancers has been associated with development and aggressiveness of cancers: PBR was shown to be a negative prognostic marker in stage III colorectal cancer (Maaser et al, 2002). In astrocytoma, PBR expression correlated with the grade of malignancy (Miettinen et al, 1995). The aggressiveness of breast cancer cells correlated not only with the extent of PBR expression but also with its nuclear or perinuclear localization (Hardwick et al, 1999).

The fact that PBR was found to be overexpressed in a variety of cancers, not only within the gastrointestinal tract but also in other tumors including breast, brain, ovary, prostate, and lung cancer (Katz et al, 1990a, 1990b; Cornu et al, 1992; Miettinen et al, 1995; Beinlich et al, 1999; Hardwick et al, 1999; Maaser et al, 2002; Sutter et al, 2002; Han et al, 2003), indicates that PBR up-regulation is a common feature during malignant transformation. Nevertheless it is noteworthy that in tissues with a relatively high basal PBR expression such as normal adrenal and normal testis, PBR expression tends to be decreased in the respective cancers (Han et al, 2003).

PBR was shown to be highly expressed in gastric mucosa (Bribes et al, 2004; Ostuni et al, 2004). Recent data demonstrate that in gastric mucosa PBR expression was functionally coupled to Ca²⁺-dependent but H⁺-independent chloride secretion possibly involved in gastric mucosa protection (Ostuni et al, 2004).

The molecular mechanisms of PBR overexpression have rarely been investigated in neoplasms yet. An important factor may be gene amplification, since it was shown that the PBR gene was amplified in a highly PBR expressing, aggressive breast cancer cell line but not so in a non-aggressive cell line that contained low levels of PBR (Hardwick et al, 2002). Moreover, differences in the expression of transcription factors and usage of promoters have recently been shown for steroidogenic and non-steroidogenic cell lines expressing different levels of PBR (Giatzakis and Papadopoulos, 2004). However, it is not known yet if these mechanisms are involved in PBR overexpression by gastrointestinal cancers.

Whether PBR overexpression is the cause or the consequence of malignant transformation has yet to be elucidated. However, its overexpression in various cancers points to the growth regulating properties which have been attributed to PBR. PBR has been associated with apoptotic and mitotic processes. It was shown that transfection-induced PBR overexpression protected lymphocytes against UV-induced apoptosis (Stoebner et al, 2001). The ability of breast cancer cells to grow in SCID mice correlated with PBR expression (Hardwick et al, 2001). In different glioma cell lines it was recently shown that PBR density correlated positively with the proliferation rate but inversely with spontaneous apoptosis (Veenman et al, 2004). PBR¢s proliferative and/or apoptosis-protective effects probably contribute to the malignant transformation during carcinogenesis.

The abundant PBR overexpression in a variety of cancers qualifies PBR as a target for diagnostic approaches, as a prognostic marker, and as a promising novel therapeutic target.

III. Diagnostic and prognostic value of PBR

PBR overexpression proved to be of prognostic relevance in colorectal and breast cancer. In UICC III colorectal cancers a high PBR overexpression correlated with a poor survival of the patients (Maaser et al, 2002). Thus, it will be interesting to investigate the prognostic relevance of PBR overexpression in other gastrointestinal cancers whose tumor biology differs from colorectal carcinoma.

The prognostic value of PBR overexpression is not limited to colorectal cancers. Recently, it was shown for breast cancer that a high PBR expression level was significantly correlated with a shorter disease-free survival in lymph node-negative patients (Galiegue et al, 2004). In astrocytoma, the extent of PBR expression correlated with the grade of malignancy and therefore with the survival of the patients (Miettinen et al, 1995).

The suitability and efficacy of PBR overexpression to detect gastrointestinal malignancies has not been investigated yet. However, PBR-based imaging by positron emission tomography (PET) has been widely studied for neuroinflammatory and neurodegenerative diseases and the use of radio-labeled PBR-specific ligands has been well established (Starosta-Rubinstein et al, 1987; Junck et al, 1989; Banati et al, 2000; Henkel et al, 2004). Both, in colorectal carcinogenesis and tumor spread PBR is frequently up-regulated (Maaser et al, 2002 and unpublished data). The surrounding tissues of colorectal primary carcinomas and of most metastases express PBR at much lower levels than colorectal cancer cells. This indicates that PBR-based imaging might well identify residual cancer tissues or micrometastases. Therefore, it is promising to apply radio-labeled PBR ligands to detect residual cancer cells or micrometastases.

Another diagnostic approach does not rely on the PBR itself, but involves its endogenous ligand diazepam binding inhibitor (DBI). Venturini et al, (1998) showed that the DBI blood level was increased in patients with HCC and recommended the blood DBI level as a marker of hepatocellular carcinogenesis.

Taken together, these data suggest that PBR qualifies as a target for both diagnostic and /or prognostic purposes in clinical gastrointestinal oncology.

IV. PBR-based therapeutic approaches

The abundant PBR overexpression in colorectal and other cancers qualifies PBR as a target for tumor-specific therapies. There are different PBR-based therapeutic approaches that warrant testing.

Many photosensitizing agents used for photodynamic therapy are structurally related to protoporphyrin IX, an endogenous ligand of PBR. Sensitivities of tumor cell lines to photodynamic therapy with porphyrins correlate positively with their densities of PBR, suggesting that porphyrin-based photodynamic therapy is mediated by PBR (Verma et al, 1998). Photodynamic therapy has already been successfully applied in the treatment of BarrettΪs mucosa as well as for dysplasia and early cancer of the esophagus (Prosst et al, 2003).

A direct approach to PBR-based therapy might involve the modulation of PBR expression. Following the hypothesis that PBR displays antiapoptotic and proliferative properties, a depletion of PBR expression might lead to apoptosis or abrogation of cell division. Papadopoulos and co-workers, (2000) showed that PBR expression can be reduced by ginkgolide B, a component of a Ginkgo biloba leaf extract. The decrease of PBR expression was associated with reduced cell proliferation and reduced tumor weight of xenografts implanted in nude mice.

Moreover, the high PBR expression in cancer could be used to direct chemotherapeutics to neoplastic tissues and thereby to increase the tumor specificity of chemotherapeutics. Enhanced cytotoxicity and increased tumor selectivity were shown for melphalan or gemcitabine in brain tumors, when these agents were coupled to synthetic PBR ligands (Kupczyk-Subotkowska et al, 1997; Guo et al, 2001).

In addition to the use of PBR ligands as a vehicle for chemotherapeutics, the ligands were shown to have anti-neoplastic potential themselves. The synthetic PBR ligands were shown to directly inhibit proliferation or to sensitize cancer cells to cytostatics. Synthetic PBR ligands such as PK 11195, FGIN-1-27, and Ro5-4864 induced apoptosis and cell cycle arrest in colorectal and esophageal cancer cells (Maaser et al, 2001; Sutter et al, 2002), in hepatocellular carcinoma cells (Sutter et al, 2004b), as well as in several other tumor entities including breast, melanoma, testis, and astrocytoma (Garnier et al, 1993; Neary et al, 1995; Landau et al, 1998; Beinlich et al, 1999; Carmel et al, 1999). Several studies showed indirect antineoplastic effects of PBR-specific ligands demonstrating that PBR ligands increased apoptosis induced by other chemotherapeutics. In hepatocellular carcinoma PK 11195 and FGIN-1-27 enhanced the chemosensitivity to paclitaxel, docetaxel, doxorubicin, and the Bcl-2 inhibitor HA14-1 (Sutter et al, 2004b). Similar sensitizing effects were observed by the use of anti-CD95, lonidamine, gemtuzumab ozogamicin, etoposide, doxorubicin, or arsenite in different tumor entities (Ravagnan et al, 1999; Decaudin et al, 2002; Walter et al, 2004).

The PBR specificity of these exogenous PBR ligands was shown using structural analogues with no affinity to PBR, which did not affect proliferation, apoptosis, or cell cycle regulation (Maaser et al, 2001; Sutter et al, 2002, 2004b). Nevertheless, the specificity of PBR ligands is discussed controversially since their antiproliferative effects are induced at micromolar ligand concentrations only thereby far exceeding the nanomolar binding affinities. However, several factors like the cellular uptake of the ligands (Sutter et al, 2002), ligand absorption to the serum of the culture medium (Lockhart et al, 2003), or different states of PBR may be responsible for the discrepancy. Kletsas et al recently showed that the antiproliferative effects of PBR ligands were independent of the extent of PBR expression in fibroblast and fibrosarcoma cells suggesting that in these cells PBR ligands target other structures than PBR (Kletsas et al, 2004).

As lipophilic agents, synthetic PBR ligands are mainly metabolized in the liver. Diazepam, which also binds to PBR with intermediate affinity, is known to have hepatotoxic side effects. Therefore possible hepatotoxic effects of synthetic PBR ligands have to be investigated. PBR ligands such as Ro5-4864 and PK 11195 were shown to inhibit protoporphyrin IX uptake, suggesting that ligand binding to PBR antagonizes its function in tetrapyrrole transport, possibly leading to cytotoxicity in long-term treatments (Wendler et al, 2003). However, PBR ligands have been safely administered in vivo without short-term toxicity and being well tolerated (Decaudin et al, 2002; Walter et al, 2004). In line with these results, no acute toxicity was observed in rat hepatocytes (Fischer et al, 2001). Nevertheless, long-term effects of PBR ligands should be addressed in future studies.

Thus, the studies presented suggest that the model of using PBR ligands is a promising approach in gastrointestinal oncology. Yet it is necessary to understand the exact mechanisms and signaling pathways of PBR ligand action to build a rational base for the use of PBR ligands. Future approaches may include the use of existing or new PBR ligands alone or in combination with other anti-neoplastic drugs.

V. Signaling pathways modulated by PBR ligands

A crucial step in the apoptotic process is the opening of the permeability transition pore (PTP) in the mitochondrial membrane. The PBR is located in the outer mitochondrial membrane being associated with the PTP. This localization suggests that PBR ligands induce apoptosis by affecting the PTP. In fact, it was shown that PBR ligands induce swelling of mitochondria and a decrease of the mitochondrial membrane potential (Maaser et al, 2001; Sutter et al, 2002). The functional relevance of PTP opening in PBR ligand-induced apoptosis was shown by using cyclosporin A. Cyclosporin A is known to inhibit the opening of the PTP and was shown to prevent PBR ligand-induced decrease of the mitochondrial membrane potential and subsequent apoptosis (Sutter et al, 2002). However, it still remains to be investigated whether PBR ligands directly affect the PTP, e.g. by changing the conformation of the PBR, or by homogeneous or heterogeneous protein interactions, or if additional signaling pathways are required. Moreover, the PTP is under the control of proteins of the Bcl-2 family. A functional interaction of PBR with the anti-apoptotic protein Bcl-2 has been suggested (Marchetti et al, 1996). In line with this, PBR ligand-induced apoptosis was shown to be associated with a decrease of Bcl-2 expression in hepatocellular carcinoma cells (Sutter et al, 2004b) and hepatic stellate cells (Fischer et al, 2001). However, in other tissues, such as pancreatic islet cells, Bcl-2 expression was not affected by PBR ligand treatment (Marselli et al, 2004). Other pathways induced by PBR ligands involve the p38 mitogen-activated protein kinase (p38MAPKinase) pathway. PBR ligands were shown to activate the p38MAPKinase, partly via caspase-3 activation, leading to overexpression of growth arrest and DNA damage-inducible protein (gadd) 45 and gadd153, apoptosis and cell cycle arrest (Sutter et al, 2003). In addition, PBR ligands were shown to modulate the extracellular signal-regulated kinase (ERK) 1/2, though the mode and time course of ERK1/2 modulation seems to be cell-type specific. In esophageal cancer cells, PBR ligands transiently activated ERK1/2 (Sutter et al, 2004a), whereas in colorectal cancer cells ERK1/2 was inactivated (Maaser et al, 2004). However, blocking the ERK1/2 pathway enhanced the PBR ligand-induced antiproliferative effects (Sutter et al, 2004a; Maaser et al, 2004), independently of the direct effect of PBR ligands on ERK1/2 activation. The antiproliferative effects of PBR ligands involve the induction of apoptosis as well as cell cycle arrest. In esophageal and colorectal cancer cells, PBR ligands induced an arrest in the G1/G0 phase of the cell cycle which occurred due to an increased expression of the cell cycle inhibitors p21^Cip1 and p27^Kip1, and a decrease of the cyclins D1 and B (Maaser et al, 2001, 2004; Sutter et al, 2002).

Many molecules and pathways involved in PBR ligand-induced apoptosis and cell cycle regulation have already been identified, but other important interactions and signaling pathways have not yet been addressed in this context. Moreover, future studies will have to clarify which of the pathways are universal and which are cell-type specific. This knowledge will help to establish if, how, and which PBR ligands are of therapeutic value, either alone or in combination with established chemotherapeutics.

VI. Concluding remarks

PBR is an attractive target for diagnostic, prognostic, and therapeutic approaches in gastrointestinal cancers. Clinical studies showed its prognostic value in colorectal carcinoma, whereas most of the therapeutic approaches have not been studied in patients yet. Therefore a series of clinical trials is required to evaluate PBR-targeted strategies in the diagnosis, prognosis, and therapy of gastrointestinal cancers.

Acknowledgements

We thank the Deutsche Forschungsgemeinschaft, Deutsche Krebshilfe, Berliner Krebshilfe, and Wilhelm-Sander-Stiftung.

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