Breast Cancer Resistance Protein in Human Tenon Fibroblasts and Its Modulation by Topical Glaucoma Medication

Abstract

Abstract: : Purpose: Multidrug resistance (MDR) has been implicated to play a role in the failure of complicated glaucoma surgery in which adjuvant antimetabolites are used. Breast cancer resistance protein (BRCP) is a recently isolated protein of the MDR family. It is unlike multidrug resistance protein (MRP) or p–glycoprotein (Pgp) a half–transporter and works as a drug–extrusion pump. It seems to be independent of Pgp or MRP expression. Coadministration of pantoprazole reduces the clearence of antimetabolites. Here, we sought to investigate the presence of BRCP in human tenon fibroblasts, which are thought to play a major role in scar formation after glaucoma surgery. We further investigated the influence of topical glaucoma medication on the expression of BRCP. Methods: Expression of BCRP was analyzed in surgically removed tenon tissues and in cultured tenon fibroblasts pretreated with topical glaucoma drugs by immunohistochemistry. Surgical specimens were obtained from patients undergoing squint surgery or trabeculectomy for a variety of glaucomatous disorders. All patients gave informed consent prior to surgery. Cultured human tenon fibroblasts were treated with Alphagan, Isoglaucon 1% and Latanoprost 0,005% for 4 weeks on a subtoxic level. The general immunostaining procedures were performed as described previously. RT–PCR: Total RNA was prepared by guanidium/phenol extraction from cultured human tenon fibroblasts of passage P2. The presence of BCRP was investigated by RT–PCR using specific PCR primers. Negative controls were performed by omitting RT during the first strand synthesis. Amplification products were separated on a 2% agarose gel and visualized with ethidiumbromide. Results: Specific bands for the chosen primer combinations could be detected by RT–PCR in P2 cells for BRCP. The negative controls did not display a signal. Strong staining in specimen from trabeculectomy patients could be detected while specimen from squint surgery showed no staining. All pretreated specimen showed a significantly stronger staining then the controls. Conclusions: The presence of the important MDR protein BRCP in human tenon fibroblasts and its upregulation through topical glaucoma medication has implications for future studies of antiproliferative therapeutic strategies in the inhibition of scar formation after glaucoma surgery. Especially the possibility to reduce the clearence of antimetabolites such as Mitomycin C should be a further target of investigation.

Keywords: wound healing