Inhibition of autophagy enhances the selective anti-cancer activity of tigecycline to overcome drug resistance in the treatment of chronic myeloid leukemia

Abstract

Background: Drug resistance and disease progression are still the major obstacles in the treatment of chronic myeloid leukemia (CML). Increasing researches have demonstrated that autophagy becomes activated when cancer cells are subjected to chemotherapy, which is involved in the development of drug resistance. Therefore, combining chemotherapy with inhibition of autophagy serves as a new strategy in cancer treatment. Tigecycline is an antibiotic that has received attention as an anti-cancer agent due to its inhibitory effect on mitochondrial translation. However, whether combination of tigecycline with inhibition of autophagy could overcome drug resistance in CML remains unclear.

Methods: We analyzed the biological and metabolic effect of tigecycline on CML primary cells and cell lines to investigate whether tigecycline could regulate autophagy in CML cells and whether coupling autophagy inhibition with treatment using tigecycline could affect the viabilities of drug-sensitive and drug-resistant CML cells.

Results: Tigecycline inhibited the viabilities of CML primary cells and cell lines, including those that were drug-resistant. This occurred via the inhibition of mitochondrial biogenesis and the perturbation of cell metabolism, which resulted in apoptosis. Moreover, tigecycline induced autophagy by downregulating the PI3K-AKT-mTOR pathway. Additionally, combining tigecycline use with autophagy inhibition further promoted the anti-leukemic activity of tigecycline. We also observed that the anti-leukemic effect of tigecycline is selective. This is because the drug targeted leukemic cells but not normal cells, which is because of the differences in the mitochondrial biogenesis and metabolic characterization between the two cell types.

Conclusions: Combining tigecycline use with autophagy inhibition is a promising approach for overcoming drug resistance in CML treatment.

Keywords: Autophagy; Chronic myeloid leukemia; Drug resistance; Energy metabolism; Mitochondrial biogenesis; Tigecycline.

Fig. 1

Tigecycline inhibits the proliferation of CML cells in dose- and time-dependent manners. (a, c) Viabilities of CML cell lines (K562, KBM5, and KBM5-STI) after treatment with different concentrations of tigecycline treatment in different time points. (b, d) Proliferations of primary CML cells obtained from newly diagnosed CML patients and refractory CML patients after treatment with different concentrations of tigecycline in different time points. Error Bars: SD of 3 independent experiments;* P < 0.05, **P < 0.01, ***P < 0.001

Fig. 2

Tigecycline suppresses mitochondrial biogenesis in CML cell lines and primary cells. (a) Effects of increasing concentrations of tigecycline on the protein levels of cytochrome c oxidase (Cox)-1, Cox-2, and Cox-4 in CML cell lines and primary cells. Tubulin was used as the reference protein in the western blotting. All the cells were cultured with tigecycline for 48 h before the experiments were conducted. (b) The relative mRNA levels of Cox-1, Cox-2, and Cox-4 in CML cells after treatment with tigecycline. (c) Evaluation of the mitochondrial membrane potential of tigecycline-treated CML cells using JC-1 staining and flow cytometry. Carbonyl cyanide 3-chlorophenylhydrazone (CCCP) was used as the positive control. (d) Reactive oxygen species (ROS) levels in the CML cells were measured by flow cytometry. Ctrl, control; TI, tigecycline-treated cells. *P < 0.05

Fig. 3

Tigecycline inhibits energy metabolism in CML cells. (a) Mitochondrial respiration of the CML cells was measured before and after treatment with tigecycline, and is represented by the oxygen consumption rate (OCR) curve and values. Oligo, oligomycin; A&R, antimycin A and rotenone. (b) Effect of tigecycline on glycolysis in the CML cells. Glycolysis capacities of the CML cells are shown by the extracellular acidification rate (ECAR) curves and values. Oligo, oligomycin. *P < 0.05

Fig. 4

Tigecycline causes apoptosis of CML cells by activating the cytochrome c/caspase-9/caspase-3 signaling pathway. (a) Apoptosis assay of the CML cells in response to stimulation with tigecycline. Left panel: a representative flow cytometry plots for CML cells stained with annexin V-FITC/PI-stained. Right panel: percentage apoptosis of the CML cells. Apoptosis was defined as the percentage of annexin V-positive cells. (b) Western blot analyses of cytochrome c, cleaved caspase-9, and caspase-3 protein levels. Cyto.C (Mito), cytochrome c protein in the mitochondria; Cyto.C (Cyto), cytochrome c protein in the cytoplasm. Cytochrome c oxidase-4 and β-actin were used as the reference proteins for the analyses of mitochondrial and cytoplasmic proteins, respectively. *P < 0.05

Fig. 5

Tigecycline induces autophagy of CML cells by downregulating the PI3K-ATK-mTOR signaling pathway. (a) Autophagic vacuoles were measured by transmission electron microscopy. Upper panel: autophagic vacuoles in CML cells with and without tigecycline treatment. Lower panel: amplified image of autophagic vacuoles in tigecycline-treated CML cells. (b) Confocal microscopy analysis of autophagy. Blue spots indicate nuclei stained with 4',6-diamidino-2-phenylindole (DAPI). Green spots indicate autophagic vacuoles stained with LC3B dye. (c) Western blot analysis to evaluate the levels of autophagy related protein P62 and LC3B, and mTOR and its upstream regulator AKT, and downstream sensors P70S6 and 4E-BP1

Fig. 6

Combining tigecycline use with autophagy inhibition shows synergetic cytotoxicity against CML cells. (a) Protein level of LC3B in CML cells that were treated with tigecycline and/or chloroquine (CQ). (b) Assay of apoptosis of CMLcells exposed to tigecycline, CQ, 3-methyladenine(3-MA), or their combination treatment. (c) ATG5 protein levels in KBM5 and KBM5-STI cells that were transiently transfected with siATG5 and evaluated by western blotting. (d) LC3B protein levels in CML cells with and without ATG5 knockdown were measured after treatment with tigecycline. (e) Apoptotic assay of CML cells subjected to ATG5 silence after stimulation with tigecycline. *P < 0.05

Fig. 7

Tigecycline has no obvious inhibitory effect on normal cells. (a) Death of normal bone marrow mononuclear cells exposed to tigecycline in different time points were measured by flow cytometry. (b) Western blot analysis of cleaved caspase-3 protein level in normal cells. (c) Effect of tigecycline on the oxygen consumption rate (OCR) curves and values for CML cells and normal cells. (d) Analyses of the glycolysis capacity of leukemic and normal cells were performed before and after stimulation with tigecycline. (e) The mitochondrial membrane potentials of leukemic and normal cells before and after treatment with tigecycline. (f) Mitochondrial mass of normal cells and CML cells was measured by incubating the cells with MitoTracker® Green FM dye. (g) Mitochondrial DNA (mtDNA) copy number was analyzed by QPCR. *P < 0.05