We assessed the cellular distribution and expression of ADK in epilepsy-associated primary glial brain tumors. We detected changes in ADK protein expression and function in astrocytic tumors and peritumoral cortex compared to control tissue. Additionally, ADK expression in the peritumoral cortex of glioma patients with epilepsy was significantly higher than in glioma patients without epilepsy.
To our knowledge this is the first study to describe the cellular distribution and expression of ADK in primary brain tumors. A previous study (Melani et al., 2003
) evaluated adenosine concentration in the extracellular fluid of tumor and peritumoral tissue of patients with high grade gliomas by intraoperative microdialysis. In this study the concentration of adenosine has been shown to be significantly reduced in the tumour tissue when compared to the control tissue, suggesting an altered purine metabolism in the tumor area (Melani et al., 2003
). The extracellular adenosine levels may reflect differences in ADK expression, accordingly we observed higher ADK expression in tumors compared to control, non-infiltrated, cortex. The variable expression levels observed within glial tumors may reflect differences in intra-tumoral vascular perfusion and hypoxia gradients and indeed hypoxia has been shown to down-regulate the expression of ADK in astroglial cells (Boison, 2008b
, Pignataro et al., 2008
). Interestingly, increased ADK expression and activity (compared to control cortex) was detected at the margin of the tumor and in the invasion front.
Immunocytochemical analysis showed ADK expression in tumor astrocytes with both nuclear and cytoplasmic labeling, however, expression was predominant in the cytoplasm. ADK exists in two isoforms: ADK-long and ADK-short isoforms (Cui et al., 2009
). It has been demonstrated that ADK-long is mainly localized in the nucleus and has an essential role in methylation reactions, being possibly involved in epigenetic controlling mechanisms. ADK-short, on the other hand, is cytoplasmically localized and regulates the extracellular adenosine concentrations (Boison, 2007
, Cui et al., 2009
). Therefore, the latter one is believed to be more involved in the regulation of neuronal excitability. Accordingly, several studies demonstrated that over-expression of ADK in mice resulted in a decrease in the adenosinergic tone and subsequently increased seizure activity (Fedele et al., 2005
, Pignataro et al., 2007
, Li et al., 2008a
, Li et al., 2008b
). Theofilas et al. (Theofilas et al., 2011
) showed that overexpression of the cytoplasmic ADK-short isoform alone is sufficient to evoke seizures. Furthermore, both experimental and human studies indicate that dysregulation of ADK is a common mechanism being operative in several forms of epilepsy (Aronica et al., 2011
The dysregulation of ADK in astrocytic brain tumors together with the upregulation of ADK observed in peritumoral infiltrated tissue of glioma patients with epilepsy supports the role of this enzyme in tumor-associated epilepsy. Importantly, significantly higher expression of ADK was detected in peritumoral tissue of glioma patients with epilepsy than in the peritumoral tissue of patients without epilepsy. Colocalisation with tumor markers (such as p53) support the expression in tumor astrocytes, however since ADK up-regulation has been detected in reactive astrocytes (Aronica et al., 2011
), we cannot exclude the contribution of a reactive glial cell population to the increase expression/activity observed within the peritumoral cortex.
The peritumoral region has been shown to be relevant for the generation and propagation of seizure activity (van Breemen et al., 2007
). The epileptogenicity of the peritumoral zone is supported by both functional and immunocytochemical studies, showing network alterations and revealing cytoarchitectural and neurochemical changes in the cortex resected from patients with intractable epilepsy associated with different types of glial tumors (Shamji, et al. 2009
, van Breemen et al., 2007
). The observed changes in ADK expression may additionally contribute to the epileptogenicity of this region, supporting a surgical approach that should aim to maximize simultaneous resection of both the tumour and (if possible) the peritumoral epileptic focus.
No significant correlation was found between ADK IR and duration of epilepsy in our cohort, however since our study does not focus on long-term epilepsy-associated tumors (LEATs; Luyken et al, 2003
) future investigations on a large cohort of LEATs are necessary to address the relationship between ADK expression and/or activity and duration and/or severity of epilepsy. Additional analysis of large series of tumors which could be stratified on the basis of the presence and absence of chronic epilepsy is also essential to further assess the value of ADK expression/activity as biomarker of epileptogenicity.
A key question is whether the increased ADK protein expression leads to an increase in enzymatic activity. Bardot and colleagues (Bardot et al., 1994
) evaluated purine metabolic enzyme activities and found no differences in enzyme activity of ADK between low- and high-grade tumors and tissue taken far from the tumor tissue in human patients. However, in this study the low- and high-grade tumors studied included both atrocytomas and oligogendrogliomas, and a histological characterization of the control tissue was not provided. The findings in the present study are not necessary in conflict with those observations. As discussed above, variable levels of ADK expression were observed within the tumor and particularly in GBM. However higher levels of ADK activity could be detected in astrocytoma grade III and in peritumoral cortex compared to control tissue. Future studies are required to further understand whether the different expression levels observed in GBM only reflect differences in hypoxia gradients within the tumor, or may be associated with different glioma cell phenotypes.
We acknowledge limitations to the interpretation of these results, since we analyzed the ADK activity in a small cohort of patients and we could not establish the relationship between ADK activity and epilepsy, because fresh tumor and peritumoral tissue samples from non-epileptic patients with astrocytoma were not available. Moreover, the expression patterns and regulation of adenosine receptors (A1
, and A3
) in both tumor and peritumoral areas deserves further investigation. Besides epileptogenesis, ADK might also play a role in tumor growth and apoptotic cell death in astrocytoma, regulating proliferation of glial and endothelial cells, as well as the antitumor immune response trough activation of receptors expressed in both astroglial and microglial cells (Abbracchio et al., 1997
, Synowitz, et al., 2006
, Dehnhardt et al., 2007
, Gessi et al. 2010
, Gessi et al., 2011
). Interestingly, increased ADK expression based on quantitative real-time PCR data was also found in human cancer samples outside the brain, such as in colorectal cancer (Giglioni, et al. 2008
). It was further demonstrated that extracellular adenosine reduced the viability of cultured astrocytoma cells (Sai, et al. 2006
), suggesting that overexpression of ADK might be a strategy of tumor cells to improve survival capabilities.
In conclusion, this study provides information on the cellular distribution and expression of ADK in primary brain tumors, suggesting a dysregulation of ADK in astrocytic brain tumors, as well as a potential involvement in the epileptogenicity of these tumors. Further understanding of the role of adenosine dysfunction and ADK in tumor-associated epilepsies requires the development of suitable animal models displaying both the clinical manifestations and neurochemical changes similar to those observed in human cerebral tumors. Since Inhibiting ADK has proven to be an effective therapy for epilepsy in different animal models (Boison, 2010
), the use of appropriate experimental models of tumor-associated epileptogenesis is essential to evaluate the possible use of adenosine augmentation therapies in patients with brain tumors and epilepsy.
Consequently, adenosine-augmenting therapeutic strategies might combine antiproliferative effects with the well-known anticonvulsive effects of adenosine.