Pivotal role of mitophagy in response of acute myelogenous leukemia to a ceramide-tamoxifen-containing drug regimen

Abstract

Acute myelogenous leukemia (AML) is a hematological malignancy marked by the accumulation of large numbers of immature myeloblasts in bone marrow. The overall prognosis in AML is poor; hence, there is a pressing need to improve treatment. Although the sphingolipid (SL) ceramide demonstrates known cancer suppressor properties, it's mechanism of action is multifaceted. Our studies in leukemia and other cancers have demonstrated that when combined with the antiestrogen, tamoxifen, the apoptosis-inducting effect of ceramide is greatly enhanced. The goal of the present study was to establish whether a ceramide-tamoxifen regimen also affects autophagic-driven cellular responses in leukemia. Using the human AML cell line KG-1, we demonstrate that, unlike exposure to the single agents, combination C6-ceramide-tamoxifen upregulated LC3-II expression, inhibited the mTOR signaling pathway, and synergistically induced KG-1 cell death in an Atg5-dependent manner. In addition, colocalization of autophagosome and mitochondria, indicative of mitophagosome formation and mitophagy, was observed. Versatility of the drug regimen was confirmed by experiments in MV4-11 cells, a FLT3-ITD AML mutant. These results indicate that the C6-ceramide-tamoxifen regimen plays a pivotal role inducing autophagy in AML, and thus constitutes a novel therapeutic design.

Keywords: Autophagy; Ceramide; Leukemia; Mitophagy; Sphingolipids; Tamoxifen.

Fig. 1.. Effect of single agent and co-administration of C6-ceramide and tamoxifen on induction of autophagy in KG-1 cells

A) Cells were exposed to combination C6-ceramide-tamoxifen for 24 h, after which the presence of autophagosomes (noted by the right-shift in fluorescence intensity) was determined using Cyto-ID and flow cytometry. B) Cells were exposed to agents indicated for 24 h. Autophagic vesicles were detected by Cyto-ID and fluorescence microscopy. C) Effect of C6-ceramide and tamoxifen on LC3-II, p62/SQSTM1 (p62), and Beclin-1 expression. Cells were exposed to the agents shown for 24 h. Levels of target proteins were analyzed by Western blot. D) Time course for expression of LC3-II, p62, and Beclin-1 in response to combination C6-ceramide-tamoxifen exposure. Autophagic activity factor (AAF). AAF = 100 × (MFI _treated − MFI _control)/MFI _treated. Mean Fluorescence Intensity (MFI).

Fig. 2.. Effect of co-administration of C6-ceramide and tamoxifen on induction of autophagic flux in KG-1 cells

A) Impact of treatment on expression of LC3-II, p62, and Beclin-1. Cells were treated with C6-ceramide-tamoxifen for 18 h and then assessed for LC3-II, p62, and Beclin-1 expression by Western blot. B) Densitometric quantitation using ratios of LC3-II, p62, Beclin-1, to β-actin. C) Effect of treatment regimen on fusion of lysosome with autophagosome. MEF cells transfected with lysosomal marker LAMP1-RFP (red fluorescent protein) were used. After treatment (18 h), MEF LAMP1^+/+ cells were stained with Cyo-ID to identify autophagosomes. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)

Fig. 3.. Effect of C6-ceramide and tamoxifen on the mTOR signaling pathway in KG-1 cells

A) Effect of single and combination agents on p-mTOR, p-70S6K (p-S6K), and p-AMPK expression. KG-1 cells were exposed to the agents shown for 24 h, after which protein expression was determined by Western blot. B) Effect of C6-ceramide-tamoxifen exposure time on expression of p-mTOR, p-S6K, and p-AKT. KG-1 cells were treated with the drug combination for the times shown. C) Densitometric quantitation of p-AKT and p-mTOR from Western blot in B.

Fig. 4.. C6-ceramide-tamoxifen combination induces mitophagy

A) Effect of drug mix on co-localization of autophagosome and mitochondria. KG-1 cells were exposed to DMSO (control) or to the drug mix (18 h), after which, cells were stained with Cyto-ID and Mitotracker Red. The fluorescence photomicroscopy overlay denotes co-incidental staining between autophagosome and mitochondria in cells exposed to mix (C6-ceramide-tamoxifen). B) GFP-LC3-MEF cells were exposed to vehicle control (DMSO) or the drug mix for 18 h, followed by staining with Mitotracker Red and Hoechst (nucleus staining). Images were obtained by fluorescence photomicroscopy. The mix overlay denotes co-localization of GFP-LC3 with mitochondria. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)

Fig. 5.. Atg5 expression impacts sensitivity to C6-ceramide-tamoxifen

KG-1 wild-type and Atg5-knockdown, KG-1-KD, were seeded in 96-well plates (50,000 cells/well). After 2 h equilibration in a tissue culture incubator, cells were treated as indicated in the figure, for 24 h. Viability was determined using alamarBlue.

Fig. 6.

C6-ceramide-tamoxifen regimen induces mitophagy and is synergistic in FLT3-ITD_mut MV4–11 cells. A) Effect of drug combination and chloroquine on autophagic vesicle formation in MV4–11 cells. Cells were seeded into 6-well plates as described Materials and methods and exposed to combination C6-ceramide-tamoxifen (10 and 5 μM, respectively) for 18 h, washed and stained with Cyto-ID and immediately imaged. Chloroquine (CQ) was used at a concentration of 25 μM. B) Western blot depicting effect of single agents and mix on expression of LC3-II and p62, and the effect of chloroquine. Cells were treated with C6-ceramide, tamoxifen, and CQ as in A. C) Evaluation of mitophagy by co-localization of autophagosome and mitochondria. Cells were exposed to DMSO vehicle (control) or the mix (10 μM C6-ceramide, 5 μM tamoxifen) for 18 h, and stained as indicated. D) Impact of combinatorial dosage on cell viability. Cells were treated with the single agents (o, tamoxifen; ▲, C6-ceramide) or the combinations (•) indicated for 18 h, and viability was assessed by alamarBlue assay. y-axis refers to fraction of cells affected (Fa), where 1.0 is equal to 100%. E) Synergy plots for the drug combination. CI indicates combination index. The lower the CI, the higher the synergy, as shown by increase in fraction affected (Fa). All doses are in μM.