D. Joshua Cameron and Dr. Heidi Vollmer-Snarr, Chemistry and Biochemistry
A2E is implicated in the etiology of AMD. We have found significant concentrations of A2E present in both lipofuscin and melanolipofuscin of human retinal pigment epithelial (RPE) cells, meaning that A2E is likely more involved in AMD than originally thought. Evidence shows that A2E, in small concentrations, is not cytotoxic alone, but becomes toxic when exposed to blue light. The light mechanism involved in A2E toxicity can be controlled and used to trigger cytotoxicity in cancer cell lines. The implications of A2E in AMD and the applications of A2E’s light triggered cytotoxicity as a possible chemotherapeutic treatment were further elucidated in this study.
The light triggered mechanism of A2E is explained in Figure 1. Controlling this mechanism is integral in our experiments. Because of A2E’s sensitivity to blue light, all work is done in the dark with only red light conditions, similar to a dark room for photography. In the human eye, A2E exists at a very low concentration, but can be very damaging as it accumulates and is oxidized to a more excited state.
The first part of my project focused on calculating the amount of A2E in the RPE and various parts within this layer of cells in human eye tissue. We found that RPE in general have about 5.86ug of A2E per eye and that most of this is contained in lipofuscin and melanolipofuscin granules deposited within the RPE cell layer. Of these two areas, melanolipofuscin had a slightly larger quantity. Washing the samples in phosphate buffered saline (PBS) prior to extraction of A2E appears to reduce the overall yield. (Figure 2)
The second aspect of my project was to utilize the light-induced oxidation of A2E as a mechanism for selectively killing cancer cells. (The initial goal was to use both MCF-7 breast caner cells as well as HL-60 leukemia cells. Unfortunately, the MCF-7 cells had maintenance problems and so only HL-60 cells were studied.) The basic idea was to grow the cells with a non-toxic concentration of A2E and then expose the cells to intense blue light and measure the affects after exposure. Using only 10uM concentrations of A2E we were able to see 100% cell death after light exposure, while seeing no such decrease in control cells either receiving A2E and no light or light and no A2E. (Figure 3)
In summary, A2E is prevalent in the human RPE and is concentrated in lipofuscin and melanolipofuscin. Care is required in preparing dissected samples as A2E can be lost during washings. Because the levels of A2E are more accurate and higher in our study, its relationship to AMD becomes stronger. A2E is also successful in treating human leukemia cells and further cell-line and in vivo studies are warranted. Results from this study have been presented at the American Chemical Society (ACS) National Meeting, Anaheim CA 2004, the ACS Regional Meeting, Logan UT, 2004, and the BYU Spring Research Conference and manuscripts are being submitted to the Journal of Biological Chemistry and the Journal of Photochemistry and Photobiology for publication.