Autophagy and Apoptosis Are Induced Simultaneously in Leukemia Cells by Vitamin K2.

Autophagy is a membrane trafficking process that leads to degradation of cytosolic proteins and organelles by lysosomes, and it is conserved throughout evolution. Autophagy is induced above basal levels in response to nutrient deprivation or trophic factor withdrawal, and it sustains metabolism through the targeted catabolism of long-lived proteins. Thus, autophagy acts as a self-limited survival mechanism. However, recent findings have suggested that autophagy is also as a potential contributor to programmed cell death.

We previously reported that vitamin K2 (VK2) selectively and potently induces the apoptosis of cultured acute myeloid leukemia cells (AML) and AML cell lines, but has almost no effect on normal hematopoietic cells. In addition, the clinical benefit of using VK2 for patients with AML and myelodysplastic syndrome has been reported. In the present study, we focused on whether autophagy was induced in AML cells by treatment with VK2.

HL-60bcl-2 cells with stable transfection of the bcl-2 gene showed 5-fold greater expression of BCL-2 compared with HL-60neo cells, and these cells were resistant to apoptosis induced by VK2, along with the occurrence of monocytic differentiation via G1 arrest. This suggested that VK2 had a differentiation-inducing effect on AML cells that were resistant to apoptosis. However, a cytocidal effect was still detectable in a caspase-independent manner. Treatment of HL-60bcl-2 cells with VK2 resulted in an increase of acid vesicular organelles (AVO) detected by acridine orange staining (for lysosomes).

Immunonoblotting with an antibody for microtubule-associated protein-1 light chain-3 (LC3) revealed the conversion of LC3-I into LC3-II a form that migrates more rapidly on SDS-PAGE and participates in the formation of autophagosomes (one of the hallmarks of autophagy). Electron microscopy revealed an increase of autophagosome formation after exposure to VK2. Finally, the induction of caspase-independent cell death by VK2 was suppressed in the presence of 3-methyladenine. These results suggest that autophagic death of HL-60bcl-2 cells was induced by VK2. Furthermore, detailed electron microscopic assessment of HL-60neo cells during exposure to VK2 revealed that autophagosome formation was more prominent compared with that occurring in H-60bcl-2 cells, although nuclear chromatin condensation was also observed at the same time. These findings indicated a mixture of the morphologic features of apoptosis and autophagy. The present results suggest that autophagy and apoptosis can both be induced simultaneously by VK2. However, autophagy only became detectable when cells were protected from rapid apoptotic death.