Induction of Autophagy in Y79 Retinoblastoma by 1,2,3,4 Tetrahydroisoquinoline (EDL-155)

Abstract

Purpose: : To study the mechanism of action of 1,2,3,4 tetrahydroisoquinoline (EDl-155) in the killing of cultured Y79 retinoblastoma cells.

Methods: : We have shown that EDL-155 kills Y-79 cells with an EC₅₀ of 12.5 µM. Preliminary observation indicated that the primary target may be mitochondria. We used Y-79 cells transfected with Luciferase to examine ATP production in a dose response and time dependent manner. Electron microscopic examination was used to examine the ultrastructure of EDL-155 treated cells. Acridine orange staining was used as an indicator of autophagy, along with an immunoblot analysis of LC-3 where a shift in LC-3 molecular weight is indicative of LC-3 incorporation into autophagasome vesicles.

Results: : When cultured Y79 cells were examined at the electron microscopic level, EDL-155 caused a dramatic decrease in the number of normal mitochondria. In addition, autophagosomes were observed wrapped in double membranes, a hallmark of autophagy. This data indicated that the destruction of the mitochondria was the primary target of EDL-155 and that the destruction of mitochondria may send the cells into terminal autophagy. To examine the effects of EDL-155 on mitochondria we examined Y-79 cells transfected with Luciferase and found that at high doses ATP production was eliminated within 4 hours of treatment. To further define the mechanism of cell death, acridine orange staining of cells treated with EDL-155 demonstrated the accumulation of orange vacuoles within the Y79 cells over time after treatment. This was not observed in control cultures. Immunoblot analysis of cells treated with EDL-155 showed a gradual shift in molecular weight of LC-3, from LC-3 I to LC-3 II (a characteristic of autophagy).

Conclusions: : Based on three indecent criteria (electron microscopy, acridine orange staining and the change in molecular weight of LC-3) EDL-155 appears to induce cell death in cultured Y-79 cells by autophagy (programmed cell death type 1).

Keywords: apoptosis/cell death • retinoblastoma • mitochondria