All-Trans-Retinoic Acid (ATRA) Induces Autophagy in Acute Promyelocytic Leukemia Cells.

All-trans-retinoic acid (ATRA), a naturally occurring derivative of vitamin A (retinol), is a potent inducer of cellular differentiation, growth arrest, and apoptosis in various tumor cell lines. ATRA induces terminal differentiation of immature leukemic promyelocytes into normal mature granulocytes in vitro and in vivo, leading to a complete remission in majority of acute promyelocytic leukemia (APL) patients. Thus, this system provides an excellent in vitro model for studying the molecular events taking place during the terminal differentiation of myeloid cells. Apoptosis (type I) and autophagy (type II) are both highly regulated forms of programmed cell death and play crucial roles in such physiological processes as the development, homeostasis and elimination of unwanted cells. In contrast to apoptosis, autophagic cell death is caspase-independent and does not involve classic DNA laddering. Recent studies suggest that cancer cells in general tend to undergo less autophagy than their normal counterparts, supporting the contention that defective autophagic cell death plays a role in the process of carcinogenesis. The present study provides the first evidence that ATRA induces autophagy in acute promyelocytic leukemia cells. We demonstrated that treatment of APL (NB4) cells with ATRA resulted in a marked increase in formation of the acidic vesicular organelles demonstrated by acridine orange staining detected by flow cytometry. ATRA treatment caused about 12-fold increase in number of acridine orange positive cells compared to untreated group. Furthermore, ATRA induced marked upregulation of microtubule-associated protein light chain 3 (LC3-II), an important hallmark of autophagy, by western blot analysis. To determine the mechanim by which ATRA induces autophagy we examined expression of Beclin-1, an autophagy-inducing protein, and Bcl-2, an inhibitor of Beclin-1 as well as levels of mTOR, a suppressor of autophagy, upon ATRA treatment. ATRA inhibited Bcl-2, upregulated Beclin-1 expression and reduced induction of activation/phosphorylation of mTOR in NB4 cells. However, rapamycin, a specific inhibitor of mTOR failed to induce autophagy, suggesting that ATRA-mediated autophagy was independent of the mTOR pathway. We are currently testing whether inhibition of Beclin-1 can prevent ATRA-induced autophagy in APL cells. In conclusion, our results revealed that ATRA plays a role in regulation of autophagy by reducing Bcl-2 and inducing protein Beclin-1, which in turn induces autophagy, suggesting a novel action and mechanism of ATRA in regulation of autophagy in APL cells.