Autophagy Induction of Acute Myeloid Leukemia (AML) Cells after Treatment with Low Dose-Cytarabine (Ara-C) and Enhanced Cytocidal Effect on AML Cells by Combined Treatment with Ara-C Plus Lenograstim.

Autophagy fundamentally refers to a process where cells enclose their own cell organelles with isolated membrane and deliver them to lysosomes for degradation, servicing as a nonspecific degradation mechanism of autologous proteins. Autophagy is induced above basal levels in response to nutrient deprivation or trophic factor withdrawal to sustain metabolism by producing catabolites. In this context, autophagy acts as a cytoprotective mechanism for cells to protect themselves from extracellular stresses. However, in a culture using cells in which autophagy-related genes were suppressed by siRNA and to which an autophagy inhibitor, 3- methyladenine (3-MA), was added, cytotoxic effects of some anticancer agents on cancer cells were diminished. Based on these results, the concept of caspase-independent cell death via autophagy, which is designated as type II programmed cell death (PCD) in contrast to apoptosis as type I PCD, has been receiving increased attention. We here report that treatment of AML cells with low does-Ara-C induces autophagy and enhances its cytocidal effect on AML cells by combined treatment with G-CSF. Treatment of AML cell line U937 cells with 1 to 50 ng/ml of Ara-C induced cell growth inhibition in a dose-dependent manner. Exposure to the higher concentrations of Ara-C (more than 10 ng/ml) induced apoptosis in U937 cells along with formation of apoptotic bodies and caspase-3 activation. However, treatment with the lower concentration of Ara-C (2.5 to 7.5 ng/ml) induced autophagy as assessed by electron microcopy and the punctuated distribution pattern of cytoplasmic microtubule-associated protein 1 light chain 3B (LC3B) by fluorescent immunocytochemistry. Low dose-Ara-C inducing -cell death and -autophagy both were suppressed when the cells were cultured in the presence of 3-MA. This suggests that the autophagic cell death but not the cytoprotective autophagy was induced in response to Ara-C. Combined treatment with G-CSF (lenograstim; 100 ng/ml) plus 2.5 to 7.5 ng/ml of Ara-C, but not plus higher concentrations of Ara-C, resulted in the enhanced cytocidal effect in U937 cells in vitro. These data indicate that combined treatment of Ara-C plus G-CSF has a therapeutic benefit for the AML patients by enhanced cytocidal effect which appears to be mediated via induction of autophagy. In addition, our data appear to explain in part the high response rate of CAG therapy in refractory AML, which consists of low-dose Ara-C in combination with G-CSF and aclarubicin.