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Summary: Purpose: Recent evidence has been obtained that the major vault protein (MVP) may play a role in multidrug resistance (MDR). We investigated the expression and cellular localization of MVP in gangliogliomas (GGs), which are increasingly recognized causes of chronic pharmacoresistant epilepsy.
Methods: Surgical tumor specimens (n = 30), as well as peritumoral and control brain tissues, were examined for the cellular distribution pattern of MVP with immunocytochemistry. Western blot analysis showed a consistent increase in MVP expression in GGs compared with that in control cortex.
Results: In normal brain, MVP expression was below detection in glial and neuronal cells, and only low immunoreactivity (IR) levels were detected in blood vessels. MVP expression was observed in the neuronal component of 30 of 30 GGs and in a population of tumor glial cells. In the majority of the tumors, strong MVP IR was found in lesional vessels. Perilesional regions did not show increased staining in vessels or in neuronal and glial cells compared with normal cortex. However, expression of MVP was detected in the hippocampus in cases with dual pathology.
Conclusions: The increased expression of MVP in GGs is another example of an MDR-related protein that is upregulated in patients with refractory epilepsy. Further research is necessary to investigate whether it could play role in the mechanisms underlying drug resistance in chronic human epilepsy.
Gangliogliomas (GGs) are the most common tumor type in young patients with chronic focal intractable epilepsy (1–3). Although they may occur throughout the central nervous system, the temporal lobe is the most common location. GGs consist of a mixture of glial and neuronal elements. This histologic composition, which also is a prominent feature of glioneuronal hamartias, has attracted considerable interest with respect to the origin, as well as the high epileptogenicity of these lesions (2,3,4–6). A maldevelopmental nature has been proposed for this tumor entity (3,7–9).
Resistance to pharmacologic treatment with a broad range of antiepileptic drugs (AEDs) is another characteristic of these lesions (2,10–12). The basis of this multidrug resistance (MDR) is still elusive, but is likely to be multifactorial, involving several nonspecific mechanisms responsible for different types of clinical drug resistance, for example, as seen with drug resistance to cytostatic drugs in cancer treatment. One possible mechanism to account for a broad medical intractability, involving AEDs with different actions, is inadequate drug concentration in the epileptogenic areas. In recent years, attention has been focused on multidrug transporters, such as P-glycoprotein (P-gp) and the family of MDR-associated proteins (MRPs) [reviewed in (13–15)]. We recently showed overexpression of these multidrug transporters involving glial, neuronal, and endothelial cells in surgical resection specimens from patients with both focal cortical dysplasia (FCD) and GG (16,17). However, other mechanisms, such as intracellular transport and drug sequestration into exocytotic vesicles, also may play a role in clinical drug resistance. Evidence points to subcellular particles called vaults for their role in such a mechanism (18,19).
Vaults are multimeric RNA–protein complexes, with one predominant structural component, the major vault protein (MVP). The high evolutionary conservation (20) and broad distribution of vaults (21) suggest a basic physiologic cellular function. The detection of high expression levels in tissues potentially exposed to toxins and in macrophages supports the notion that the physiologic function of these molecules is to provide protection (21,22). Vaults are localized mainly in the cytoplasm, but a small fraction also has been localized at the nuclear membrane and the nuclear pore complex (23). Thus, although vault function remains undetermined, evidence supports the role of vaults in the vesicular transport of several compounds, mediating a bidirectional nucleocytoplasmic exchange (23–27).
The functional role of MVP has increased in significance in view of the finding the MVP vaults are overexpressed in many human tumors, and a connection has been shown between their expression and MDR (21,22,26,28–32). Although a direct involvement of vaults in MDR could not be demonstrated in the MVP knockout mouse model (33), it cannot be excluded that other mechanisms of drug resistance become upregulated on disruption of MVP and that vaults might still be involved in the protection against long-term exposure to drugs. In addition, recent evidence supports a role of vaults in other cellular process, such as cellular differentiation (34).
In the present study, Western blot and immunocytochemistry with antibodies (Abs) specific for MVP were performed in surgical specimens of patients with GGs and pharmacoresistant epilepsy. Our major aim was to provide data that may help to define the expression level and cellular distribution of MVP in tumor, peritumoral, and normal brain tissue, and may provide better insights into the mechanisms underlying MDR to treatment with AEDs in patients with chronic focal epilepsy.
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- MATERIALS AND METHODS
Recent evidence suggests that the general mechanisms of resistance to drug treatment that are active in human cancer patients also may be involved in refractoriness to AEDs (48). To date, the expression pattern and the contribution of transmembrane transporter proteins (P-gp and MRP) to MDR has been widely investigated in several causes of refractory epilepsy (including GGs) (16,17). Resistance to drugs is generally considered to be multifactorial; thus additional molecules such as MVP also are believed to contribute to MDR (22). Whether they also play a role in drug resistance in patients with refractory epilepsy remains to be established. The detection of and the expression pattern of MVP in control and epileptic human brain tissue represent the first step to increase our knowledge about the functions of these proteins, including their potential role in the intrinsic MDR phenotype.
In the present study, we demonstrated that GGs, the most common tumor entity in young patients with medically intractable epilepsy, express high amounts of MVP. Gradient centrifugation analysis indicates that all MVP in GGs assembles in particles behaving as intact vaults. In agreement with previous studies in astrocytic brain tumor cells (30), MVP particles in GGs are located predominantly at cytoplasmic sites. The overexpression and the subcellular location of MVP detected via immunoblotting are confirmed by immunocytochemistry. Expression of MVP was observed in the cytoplasm of neuronal and glial cells of GG specimens. In normal brain, both neurons and resting glial cells do not have detectable expression of MVP. This observation is in agreement with previous studies showing low vault IR in adult brain tissue in both humans and rats (49,50). Whereas adult glial and neuronal cells lack any detectable expression of MVP, strong MVP expression is observed during embryonic and early postnatal development in rat brain (50). Thus reappearance of this embryonic protein in GGs might support the malformative and plastic nature of these tumors. Although the histogenesis of glioneuronal tumors still remains speculative, one possible hypothesis is that they originate from still immature or multipotent dysplastic cells. Recent studies demonstrate the detection in GGs of other proteins, which are expressed early during development and reflect an immature phenotype (3,51,52).
Activation of MVP expression also might occur during the process of malignant transformation of astrocytes. Malignant astrocytes in vitro (cell lines and primary cultures) express high amounts of MVP protein (30). Moreover, a strong expression of MVP has been reported in both tumor glial cells and neoplastic vessels of surgical specimens from patients with glioblastomas (53). Accordingly, we also observed increased expression of MVP in the vascular compartment of GGs. Expression of MVP in blood vessels has been observed during development in rat brain (50). Whether MVP is expressed exclusively by vascular endothelial cells (47) or also by perivascular pericytes (50) is still unclear. Because no single commonly used marker identifies all pericytes with certainty, it is difficult to identify pericytes in pathologic conditions, such as cancer, when these cells change their expression of marker proteins (54). Immunogold electron-microscopic studies (in non–paraffin-embedded material) will be necessary to define the cellular localization of MVP within blood vessels. Recently increased expression of other MRPs (multidrug transporter proteins, MRP1 and P-gp) was observed in the vascular compartment of glioneuronal lesions (17). This is of particular interest in relation to their function as outwardly directed efflux pumps, which may limit the brain accumulation of different lipophilic drugs, including AEDs. The different physiologic functions and the functional consequences of the increased expression of MRPs at the level of the blood–brain and blood–tumor barriers in drug-resistant human epilepsy remain to be determined.
Interestingly, the overexpression of MVP labeling was observed in the lesion and not in perilesional areas. This observation supports the hypothesis of constitutive rather than induced or acquired expression and is in keeping with the previously suggested role for the transmembrane transporter protein overexpression observed in different developmental lesions associated with refractory epilepsy (16,17). It is not likely that treatment with AEDs per se is responsible for the observed intralesional MVP overexpression, because tissue adjacent to the lesion has been theoretically exposed to the same drugs. The potential interactions of vaults with AEDs, as well as the influence of AED exposure on MVP expression, should be investigated further in normal and tumor astrocytes expressing MVP protein in vitro.
We cannot totally exclude that the overexpression in epileptogenic brain tissue is related to seizure activity. MVP expression was detectable in sclerotic hippocampus from patients with dual pathology. Association of GGs with mesial temporal sclerosis (MTS) has been previously described and may define a distinct pathophysiologic category of MTS (55,56). Strong MVP expression was observed in hypertrophic cells in the dentate hilus. Hypethrophic cells within the dentate hilus and similar overexpression of MVP also were observed in adult patients with temporal lobe epilepsy [TLE; (57) Sisodiya et al., unpublished observation]. Activation of MVP expression may represent a feature of hippocampal pathology in TLE. In our study, with fixed material, it was not possible to investigate the spatiotemporal development or the functional consequences of this overexpression. For this purpose, the use of experimental models of pharmacoresistant epilepsy is required.
Although the cellular function of vault has not been completely clarified, tissue-distribution studies point to a protective role against toxic compounds as for other MRPs [P-gp and MRP; (22)]. Overexpression of MVP [together with P-gp and MRP1; (17)] may function as a general protection mechanism, which could influence the fate and the survival of cells expressing this phenotype early during development. Moreover, overexpression of MVP in the epileptogenic neuronal component of GGs also may affect the response to AEDs, by changing the subcellular compartmentalization of drugs (22).
These results [together with previous observations in GGs (17)], indicate that drug resistance–related proteins (P-gp, MRP1, and MVP) are regulated in concert in GGs. MDR in these lesions may be the result of different mechanisms operating at different levels. Expression of drug resistance–associated proteins at the level of the blood–brain and blood–tumor barriers may impair the penetration of therapeutic agents, leading to decreased tissue drug concentrations. Moreover, activation of expression at the level of the neuronal and neuroglial epileptogenic component of the GGs may interfere with the intracellular activities of the drugs by compartmentalization of drugs away from the intracellular target (MVP) or pump molecules (P-gp, MRP1). Thus evaluation of the role of vault molecules in the sequestration and compartmentalization of AEDs might be worthwhile and requires careful consideration.