Background
Chronic myeloid leukemia (CML) is a myeloproliferative neoplasm companied with the BCR-ABL fusion gene, which is encoded by the Philadelphia chromosome [
1]. The BCR-ABL fusion protein plays a key role in CML leukemogenesis by activating its downstream signaling pathway of survival and proliferation [
2,
3]. Imatinib is a targeted competitive inhibitor of BCR-ABL tyrosine kinase that has changed the clinical treatment and prognosis of CML. Other tyrosine kinase inhibitors (TKIs) such as dasatinib and nilotinib have a higher anti-leukemic activity and are associated with fewer side effects. However, acquired resistance to TKIs is one of the main obstacles to effective CML treatment and is involved in gene amplification of ABL tyrosine kinase point mutations [
4]. Patients with these ABL tyrosine kinase point mutations generally have a poor prognosis and a higher mortality rate.
The survival, proliferation and drug resistance property of leukemic cells are mainly dependent on their crosstalk with the bone marrow microenvironment [
5]. In addition, hypoxia and nutrient deficiency are characteristics of the microenvironments of cancers [
6]. Under such hostile surroundings, leukemic cells adapt dynamically by adjusting their metabolic phenotype. In order to maintain the energy balance between ATP production and the need for excessive proliferation and survival, cancer cells develop a novel metabolic phenotype that is more dependent on glycolysis than on mitochondrial oxidative phosphorylation (OXPHOS). This phenomenon is known as Warburg effect [
7]. In contrast, some researches have recently demonstrated that mitochondrial respiration still plays a vital role in the metabolic properties of cancer cells by reinforcing mitochondrial activity [
8,
9]. In tumorigenesis, mitochondria are not only biosynthetic organelles involved in bioenergetic reactions, but they are also survival signaling mediators for the cancer cells. Therefore, understanding and targeting the mechanisms underlying mitochondrial function would be critical exploitable points in the development of cancer therapies.
Autophagy can be induced when cancer cells subjected to hypoxia, poor nutritional conditions and chemotherapy to allow cancer cells adapt to the developmental microenvironment [
10,
11]. Inhibition of tyrosine kinase by TKIs not only results in cell death but also causes the induction of autophagy in CML cells. Importantly, inhibition of autophagy by pharmacological inhibitors (chloroquine and bafilomycin A) or knockdown of the autophagy genes ATG5 and ATG7 in CML cells could potentiate TKIs-induced cell apoptosis in CML cells [
12]. These researches demonstrated that inhibition of autophagy could improve the treatment efficiency of TKIs and combining of autophagy inhibition with other anti-cancer drug may be more efficient treatment to CML cells. Currently, it has been shown that the antibiotic tigecycline has an interesting “side effect” of inhibiting mitochondrial translation in cancer cells, which results in the killing of cancer cells, as well as cancer stem cells [
13]. The observations challenged the traditional concept that cancer treating like an infectious disease with less toxicity. Based on this premise, we hypothesized that tigecycline could be used as an anticancer drug, as it induces significant apoptosis of CML cells companied with autophagy. Moreover, inhibition of autophagy could enhance the anti-leukemic effect of tigecycline and reverse drug resistance to TKI therapy which mainly attributable to ABL tyrosine kinase point mutations.
Methods
Cell lines and cultures
BCR-ABL positive KBM5 cells and K562 cells were used as imatinib-sensitive cell lines. On the other hand, KBM5 cells with T315I mutation (KBM5-STI cells), which were provided by Prof. Michael Andreeff (Section of Molecular Hematology and Therapy, Department of Leukemia, The University of Texas M.D. Anderson Cancer Center, Houston, TX, USA), were used as an imatinib-resistant cell line. Thirteen primary CML cells were obtained from eight newly diagnosed patients (cases at chronic phase without any mutations) and five refractory patients (2 cases with T315I mutation, 3 cases without any mutations) after obtaining written informed consent. Eight healthy donors were selected by matching their ages and sex to those of the CML patients. The study was approved by the Institutional Review Board of Nanfang Hospital (Guangzhou, China).
Cell viability assay
Cell viability assay was performed using Cell Counting Kit-8 (CCK8; CK40; Dojingdo, Kumamoto, Japan). Cells were seeded in 96-well plates and then exposed to tigecycline (Hisun Pharmaceutical Company, Fuyang, China) and/or chloroquine (CQ) (14774; Cell Signaling Technology, Danvers, MA) at the indicated concentrations for 48 h. The cells were incubated with CCK-8 solution for 2 h, after which absorbance was measured at 450 nm using a SpectraMax Plus microplate reader (Molecular Devices, Sunnyvale, CA, USA).
Flow cytometry analysis
After stimulation with tigecycline and/or CQ, the cells were subjected to flow cytometry experiments. An annexin V-FITC and PI apoptosis detection kit (AD10, Dojingdo) was used for apoptosis assay according to the manufacturer’s instructions. A mitochondrial membrane potential assay kit (12664, Cell Signaling Technology, Danvers, MA, USA) was used to detect mitochondrial membrane potential in the CML cells using the fluorescent dye JC-1.When JC-1 permeates into intact mitochondria, it aggregates to orange-red fluorescence. However, JC-1 infiltrates into impaired mitochondria, the dye changed into green fluorescence. The relative ratio of the green/red fluorescence is therefore used as an indicator of mitochondrial membrane potential. The generation of reaction oxygen species (ROS) was monitored after the cells were stained with a fluorometric dye using a Fluorometric Intracellular ROS Kit (MAK142; Sigma-Aldrich, St. Louis, MO, USA) according to the manufacturer’s instructions. Furthermore, mitochondrial mass was measured using Mito Green Probe (KGMP0072, Keygen Biotech, Nanjing, China) after incubating the cells at 37 °C for 30 min. All the assays mentioned above were digitized using a FACSCalibur flow cytometer (Becton-Dickinson, Franklin Lakes, NJ, USA).
Measurement of oxygen consumption rate (OCR) and extracellular acidification rate (ECAR)
OCR and ECAR were measured using Seahorse XF24 Flux Analyzer (Seahorse Bioscience, Shanghai, China). Briefly, the cells were plated on XF24 cell culture plates coated with Cell-Tak solution (1 mg/mL; MAP-O4012; ACRO Biosystems, Newark, DE, USA). K562, KBM5, and KBM5-STI cells were seeded at 250,000 cells/well, 300,000 cells/well, and 300,000 cells/well, respectively, whereas primary CML cells were plated at 300,000 cells/well. The cells were seeded in each well in 100 μL of XF Assay Medium (102353–100, Seahorse Bioscience) supplemented with 1 mM pyruvate, 2 mM glutamine, and 10 M glucose for OCR analysis, or 1 mM glutamine for ECAR measurement. Cell Mito Stress Test Kit (103015–100, Seahorse Bioscience) and Glycolysis Stress Test Kit (103020–100, Seahorse Bioscience), which contained the relevant compounds for each assay, were used to measure OCR and ECAR, respectively. Oligomycin, carbonyl cyanide 4-(trifluoromethoxy)phenylhydrazone (FCCP), and a mixture of rotenone and antimycin A were used for the OCR analysis, whereas glucose, oligomycin, and 2-deoxyglucose (2-DG) were used for the ECAR analysis. The compounds were serially injected into the cells to measure mitochondrial respiration and glycolysis function, respectively.
Western blotting
Western blotting was performed as before [
8]. Briefly, proteins were separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), transferred to nitrocellulose membranes, and sequentially incubated overnight with primary antibodies at 4 °C. After incubation with secondary antibodies, signals were visualized using a chemiluminescent detection reagent (WBKLS0500; Millipore, Billerica, MA, USA). Before the analysis of cytochrome c, the cytosolic and mitochondrial sub-cellular fractions needed were separated using a mitochondria isolation kit (C3601; Beyotime Biotechnology, Shanghai, China). The western blotting analysis was conducted using the following primary antibodies: anti-cytochrome c (11940), anti-cleaved-caspase-9 (32539), anti-cleaved-caspase-3 (13847), anti-LC3B (51520), and anti-p62 (91526), which were obtained from Abcam (Cambridge, UK); and p-AKT (Ser473, 4060), p-AKT (Thr308, 13038), AKT (9272), p-mTOR (Ser2448, 5536), p-P70S6K (Thr389, 9234), p-4E-BP1 (Thr37/46, 2855), and anti-ATG5 (12994), which were obtained from Cell Signaling Technology; and anti-Cox-1(19998), anti-Cox-2(19999) and anti-Cox-4 (69359), which were obtained from Santa Cruz Biotechnology (California, USA).
Fluorescence and confocal microscopy
After treatment with tigecycline, CQ, or 3-methyladenine (3-MA) (9281, Sigma-Aldrich), the CML cells were prepared for the autophagy detection experiment. An autophagy detection kit (139484, Abcam) was optimized and used for the detection of autophagy in the CML cells by fluorescent scanning confocal microscopy (Olympus FV10i; Olympus, Tokyo, Japan).
Transmission electron microscopy
The cells were stained with uranyl acetate and lead citrate on a microtome (Leica Ultracut; Leica, Wetzlar, Germany) and examined with a transmission electron microscope (Hitachi H-7650; Hitachi, Tokyo, Japan) at an accelerating voltage of 60 KV. Micrographs were taken at × 7,000 or × 20,000 magnification.
Quantitative PCR (QPCR)
Mitochondrial DNAs (mtDNAs) were extracted from the fresh bone marrow mononuclear cells obtained from the CML patients and healthy donors and stored at −20 °C until use. Relative mtDNA copy number and the mRNA levels of Cox-1, Cox-2 and Cox-4 were determined by qPCR method. The primer sequences used were as follows: forward primer (Cox-1-F) 5’-CTATACCTATTATTCGGCGCATGA-3’and reverse primer (Cox-1-R) 5’- CAGCTCGGCTCGAATAAGGA-3’ for Cox-1; forward primer (Cox-2-F) 5’- CTGAACCTACGAGTACACCG-3’ and reverse primer (Cox-2-R) 5’- TTAATTCTAGGACGATGGGC-3’ for Cox-2; forward primer (Cox-4-F) 5’- GCCATGTTCTTCATCGGTTTC-3’ and reverse primer (Cox-4-R) 5’- GGCCGTACACATAGTGCTTCTG-3’ for Cox-4; forward primer (18 s-F) 5’- AGGAATTGACGGAAGGGCAC-3’ and reverse primer (18 s-R) 5’- GGACATCTAAGGGCATCACA-3’for 18S; forward primer (ND1-F) 5’-CCC TAA AAC CCG CCA CAT CT-3’ and reverse primer (ND1-R) 5’-GAG CGA TGG TGA GAG CTA AGG T-3’ for ND1; and forward primer (human globulin (HGB)-F) 5’-GTG CAC CTG ACT CCT GAG GAG A-3’ and reverse primer (HGB-R) 5’-CCT TGA TAC CAA CCT GCC CAG-3’ for HGB. QPCR reactions were performed on an ABI Prism 7900 sequence detection system (Applied Biosystems, Foster City, CA). The relative abundance of a transcript was represented by the threshold cycle of amplification (CT). The comparative CT method was calculated per the manufacturer's instructions. The expression level of Cox-1 relative to the baseline level was calculated as 2–ΔC
T
(Cox-1), where ∆CT is (average Cox-1 CT – average 18 s CT) and is CT (average CT -treated sample – average CT -untreated sample. The calculation methods of expression levels of Cox-2, Cox-4 and ND1 are the same as that of Cox-1. For the calculation method of expression level of ND1, the reference one is HGB.
Small interfering RNA transfection
Cells were transfected with siRNA (ATG5-F, 5’ CAACTTGTTTCACGCTATATCAGG; ATG5-R, 5’ CACTTTGTCAGTTACCAACGTCA) designed by Guangzhou Land Bio Co. Ltd. (Guangzhou, China) using Lipofectamine® 2000 Transfection Reagent (11668; Invitrogen, Carlsbad, CA, USA) according the manufacturer’s instructions.
Statistical analysis
GraphPad Prism 5 (GraphPad Software Inc., San Diego, CA, USA) was used to perform statistical analysis. The results have been expressed as mean ± standard deviations. Test of comparison between two groups were done using Student’s t-test (two tailed) and comparisons between multiple groups were done by one-way ANOVA. P values < 0.05 were considered statistically significant.
Discussion
Although the major breakthough TKIs make, the emergence of drug resistance, which can occur in a mutant-dependent or mutant-independent manner, is still is a challenge in their use. The potential therapeutic targets in cases of drug resistance, which are based on differences between cancer cells and normal cells, remain to be explored. In tumorigenesis, glycolysis is not only an energy-production means but also a biosynthetic tool that supports cell survival and proliferation. Mounting evidence shows that abnormal dependence on glycolysis during energy production in cancer cells is well correlated to therapeutic resistance in cancer treatment [
26]. It has been reported that upregulation of lactate dehydrogenase-A, which plays a key role in the glycolytic pathway for regulating the conversion of pyruvate to lactate, causes resistance to paclitaxel in breast cancer treatment [
27]. Furthermore, a high expression of pyruvate kinase M2 has been noted to contribute to resistance to 5-fluorouracil in patients with colorectal cancer [
28]. Additionally, a high level of pyruvate dehydrogenase is linked to sorafenib-acquired resistance in hepatocellular carcinoma cells [
29]. In the present study, the levels of glycolysis and mitochondrial respiration were higher in the primary CML cells than they were in the healthy cells. These data suggest that the CML cells relied heavily on glycolytic and mitochondrial functions to meet the high demands of energy production and metabolism and these effects were supported by an accelerated mitochondrial protein translation.
Tigecycline is a relatively new antibiotic that has a broad antibacterial spectrum against gram-positive and gram-negative pathogens via binding to the 30S bacterial ribosomal subunit. Interestingly, the 30S bacterial ribosome is homologous to the 28S mitochondrial ribosome in eukaryotic cells [
13]. Consequently, tigecycline acts as a novel anticancer drug due to its inhibition of mitochondrial protein translation. However, whether tigecycline improves drug-resistance in CML is still unknown. Hence, we implemented a comprehensive in vitro study to evaluate the mechanism underlying the anti-leukemic effect of tigecycline. First, tigecycline inhibited the growths of CML primary cells and cell lines, including imatinib-sensitive and imatinib-resistant cells, by impairing mitochondrial biogenesis. In addition, tigecycline suppressed the activities of the mitochondrial-encoded proteins Cox-1 and Cox-2, but not the activity of the nuclear-encoded protein Cox-4. This resulted in a downregulation of mitochondrial electron transport respiratory chain and a reduction in OCR. In addition, there was mitochondrial membrane depolarization and low ROS production. As a byproduct of the electron transport chain, ROS usually increase rapidly after treatment of cells with inhibitors of the electron transport chain, such as oligomycin, rotenone, and antimycin A. We speculated that tigecycline may not inhibit electron transport chain enzymes directly in a unique mechanism distinct from other electron transport chain inhibitors do. Furthermore, tigecycline reduced the enhanced glycolytic level (ECAR) in the CML cells and showed an inhibitory effect on the CML cells, including those that were imatinib-resistant with T315I mutation. Subsequently, the tigecycline-treated CML cells were prone to apoptosis via activation of the cytochrome c/caspase-9/caspase-3 pathway. Recently, Karvela and colleagues demonstrated that knockdown of the autophagy-mediated protein ATG7 leads to decreased glycolysis [
30]. Moreover, autophagy serves as a “weapon” for cancer cells to response to chemotherapy and to oppose the mechanisms of apoptosis. Therefore, we explored the role of tigecycline in autophagy in the CML cells. We observed that tigecycline induced autophagy in the CML cells by downregulating the PI3K-AKT-mTOR signaling pathway. Next, combined treatment by using tigecycline and autophagy inhibition further enhanced the sensitivity of the CML cells to tigecycline-induced cytotoxicity. Surprisingly, tigecycline showed no cytotoxicity to the normal cells because of the differences in the metabolic type and mitochondrial biogenesis between leukemic cells and their healthy counterparts. The levels of glycolysis and mitochondrial respiration in the primary CML cells were higher than the respective levels in the normal cells. Additionally, mitochondrial mass and relative mtDNA content were higher in the primary CML cells than they were in the healthy control cells.
Due to the selective anti-leukemic activity of tigecycline, the latter seems to be a promising agent for tumor treatment. However, it has been reported that, after monotherapy with tigecycline, no significant pharmacodynamic changes or clinical responses were observed in patients with refractory acute myeloid leukemia (AML) [
31]. This suggests that the administration route, dose, and use of tigecycline in combination with other treatments should be explored to improve the anticancer effect of tigecycline. In this regards, most CML patients after combination treatment of tigecycline with pharmacological inhibition of autophagy may obtain efficient treatment response, even the patients with poor prognostic markers.
Acknowledgements
We are very grateful to Prof. Michael Andreeff for the cell line KBM5-STI cells.