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Abstract Number: 509
Use of pheophorbide a, an ABCG2 specific substrate, to probe for ABCG2 expression and function
Robert W. Robey, Kenneth Steadman, Orsolya Polgar, Margaret Blayney, Prakash Mistry, Susan E. Bates. NIH/NCI, Bethesda, MD and Xenova Group, Berkshire, United Kingdom.
Pheophorbide a (PhA), a chlorophyll catabolite, was shown to be an ABCG2 substrate by Jonker et al, based on Abcg2-/- knockout mouse studies (Jonker et al., Proc. Natl. Acad. Sci. USA 99:15649-15654, 2003). Seeking a potential ABCG2-specific substrate, we developed a functional assay for ABCG2 using PhA and the ABCG2 specific inhibitor fumitremorgin C (FTC). In studies with selected cell lines expressing high levels of P-glycoprotein (Pgp), multidrug resistance associated protein 1 (MRP1), or ABCG2, PhA transport was detected only in cells expressing ABCG2 suggesting that, among the three transporters implicated in multidrug resistance, PhA is specific for ABCG2. FTC-inhibitable PhA transport was found to correlate with cell surface ABCG2 expression as measured by the anti-ABCG2 antibody 5D3. Additionally, PhA transport was found to be unaffected by the ABCG2 mutations at amino acid 482 that are known to affect substrate specificity. Using PhA as a model to screen for potential ABCG2 inhibitors, we found that 100 µM of the cyclin-dependent kinase inhibitor UCN-01 or 1 µM of the Pgp inhibitor tariquidar inhibited ABCG2-mediated PhA transport. The interaction of these two compounds with ABCG2 was confirmed in 4-day cytotoxicity assays. ABCG2-mediated resistance to SN-38 and topotecan was abrogated in ABCG2-transfected cells treated with 1 µM tariquidar, and ABCG2-transfected cells were 6- to 7-fold resistant to UCN-01. PhA is an ABCG2-specific substrate with potential value in measuring ABCG2 function and expression in clinical samples. Further studies exploring the potential of tariquidar as a multispecific blocker for Pgp and ABCG2 are warranted.
Presenter: Robert W. Robey
Affiliation: NIH/NCI, Bethesda, MD; E-mail: robeyr@mail.nih.gov
Copyright © 2004 American Association for Cancer Research. All rights reserved. Citation information: Proceedings of the AACR, Volume 45, March 2004. |
