Abstracts

Rationale: Drug resistance remains a major problem in clinical epileptology. While a role for ABC transporter polymorphisms in antiepileptic drug resistance has been suggested, every controlled study to date has failed to replicate the association (e.g., Tan et al. 2004). Recently, an inverse correlation between MDR1 SNP and drug resistance was reported (Seo et al. 2006). We recently reported a major role for the multidrug transporter RLIP76in drug resistance to antiepileptic drugs (Awasthi et al. 2005). This was challenged in a recent paper stating that RLIP76 is not localized in membrane and hence not a transporter. We have performed a number of experiments to demonstrate the validity of our report linking excessive RLIP76 expression to multiple drug resistance in epilepsy. Methods: Dual-antibody immunocytochemistry and Immunoblotting were used to confirm the expression at the tissue and subcellular levels of the transporter RLIP76. Cells and brain sections from human brain resections were used to confirm upregulation in epileptic brain. Results: RLIP76 is drug transporters expressed in vessels from the human brain, in particular from epileptics. This was shown with dual-label immunodetection and by comparing expression in epileptic vs. non-epileptic brain and is also recognized in web-based databases such as DSI, NCBI-OMIM, Pharmgkb, etc. We intentionally used the antibody employed by Soranzo and colleagues to show that regardless of the antibody used, RLIP76 expression was always identical to what reported by us in the original manuscript (Awasthi et al. 2005). RLIP76 is expressed, as previously published by us and others, on the cell membrane. This was demonstrated by dual label immunocytochemistry on cultured cells. There are three non-synonymous SNPs detected in RLIP76: we have experimentally challenged the assumption of lack of RLIP76 SNP involved in transport. We constructed the mutants of RLIP76 (Lys [K] 149 → Glu [E] 149 and Arg [R] 208 →Gln [Q] 208) and tested the transport properties of these mutants for doxorubicin. Mutant Lys [K] 149 → Glu [E] 149 shows 30% less activity than the wild type RLIP76 while there is no significant change in the activity of mutant, Arg [R] 208 →Gln [Q] 208Conclusions: We have therefore confirmed our previous findings and have produced additional results that further support a role for membrane-bound RLI76 in the extrusion of drugs. The increased expression in multiple drug resistant epileptic brain further supports a role in refractoriness to AED.