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Antibiotic ivermectin selectively induces apoptosis in chronic myeloid leukemia through inducing mitochondrial dysfunction and oxidative stress
Biochemical and Biophysical Research Communications xxx (2018) 1e7
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Antibiotic ivermectin selectively induces apoptosis in chronic myeloid leukemia through inducing mitochondrial dysfunction and oxidative stress Jiaqiao Wang, Yanhua Xu, Huihui Wan, June Hu* Department of Oncology, Jingzhou Central Hospital, The Second Clinical Medical College, Yangtze University, Jing Zhou, People’s Republic of China
a r t i c l e i n f o
a b s t r a c t
Article history: Received 5 February 2018 Accepted 7 February 2018 Available online xxx
Mitochondria has been a promising target in blood cancer given their unique dependencies on mitochondrial functions compared to normal hematopoietic cells. In line with this concept, we show that an anthelminthic drug ivermectin selectively kills chronic myeloid leukemia (CML) cells via inducing mitochondrial dysfunctions and oxidative stress. Ivermectin is significantly more effective in inducing caspase-dependent apoptosis in CML cell line K562 and primary CML CD34 than normal bone marrow (NBM) CD34 cells. Ivermectin also augments in vitro and in vivo efficacy of standard CML tyrosine kinase inhibitors. Mechanistically, ivermectin inhibits respiratory complex I activity and suppresses mitochondrial respiration in K562 and CML CD34 cells. Interestingly, we demonstrate that mitochondrial respiration are lower in NBM CD34 compared to malignant CD34 cells. In addition, ivermectin also induces mitochondrial dysfunctions in NBM CD34 cells in a similar manner as in CML CD34 cells whereas NBM CD34 cells are significantly less sensitive to ivermectin than CML CD34 cells. These suggest that NBM CD34 cells are more tolerable to mitochondrial dysfunctions than CML CD34 cells. Consistently, ivermectin induces higher levels of oxidative stress and damage in CML than normal counterparts. Antioxidant NAC rescues ivermectin's effects, confirming oxidative stress as the mechanism of its action in CML. Our work provides the fundamental evidence to repurpose ivermectin for CML treatment. Our work also highlights the therapeutic value of targeting mitochondria respiration in CML. © 2018 Elsevier Inc. All rights reserved.
Keywords: Ivermectin CML Mitochondrial respiration Oxidative damage Selectivity
1. Introduction Chronic myeloid leukemia (CML) is a hematological stem cell malignancy and characterized by the presence of Philadelphia chromosome that generates the BCR-ABL oncogene [1]. BCR-ABLpositive hematopoietic CD34 stem/progenitor cells have a proliferative advantage which eventually displace residual normal hematopoiesis [2]. Treatment of BCR-ABL tyrosine kinase inhibitors (TKIs), such as nilotinib and dasatinib, has significantly improved clinical outcomes. However, primitive leukemia CD34 stem/progenitor cells are retained even in patients achieving remission, suggesting that treatment with TKIs alone is insufficient to cure CML [3,4].
* Corresponding author. Department of Oncology, Jingzhou Central Hospital, The Second Clinical Medical College, Yangtze University, Renmin Road 1, 434020 Jing Zhou, People’s Republic of China. E-mail address: [email protected] (J. Hu).
Ivermectin is an anthelminthic drug used for the treatment of many types of parasites with unknown mechanism of action [5]. However, ivermectin has been recently identified to exhibit potent anti-cancer activities. It induces cytostatic autophagy and necrosis in breast cancer cells via blocking the PAK/Akt axis and modulating P2X4/P2X7 signaling, respectively [6,7]. Ivermectin inhibits xenograft growth of many types of solid cancer without obvious side effects via inhibiting WNT-TCF pathway [8]. It also induces chloride-dependent membrane hyperpolarization and cell death in blood cancer, such as leukemia [9]. In this study, we investigated the effect and selectivity of ivermectin in CML cell line K562, primary CML CD34 and normal bone marrow (NBM) CD34 cells. We next investigated whether ivermectin can sensitize CML cells to multiple BCR-ABL TKIs. Finally, we determined the target of ivermectin in CML cells. We are the first to demonstrate that ivermectin induces apoptosis in CML cells while sparing normal hematopoietic cells, via disrupting mitochondrial functions and inducing oxidative stress. The different dependencies of CML and normal CD34 cells to mitochondrial functions might
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Please cite this article in press as: J. Wang, et al., Antibiotic ivermectin selectively induces apoptosis in chronic myeloid leukemia through inducing mitochondrial dysfunction and oxidative stress, Biochemical and Biophysical Research Communications (2018), https://doi.org/ 10.1016/j.bbrc.2018.02.063
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contribute to their sensitivities to ivermectin treatment.
2.6. Oxidative DNA damage
2. Materials and methods
Cells were treated with drug or combination for 24 h. Cells were harvested and DNA was extracted using the DNEasy Mini Kit (Qiagen). 8-OHdG levels were quantified using the OxiSelect Oxidative DNA Damage ELISA Kit (Cell Biolabs) according to manufacture's protocol. Absorbance was read on a Spectramax M5 Microplate reader at 450 nm.
2.1. Primary cells, cell culture and drug reconstitution Human peripheral blood mononuclear cells were isolated using Ficoll separation. Primary CML CD34 cells were further purified using CD34 MicroBead isolation kit (Miltenyi Biotec, Germany) according to manufacturer's instructions. CML samples were obtained from patients seen at Jingzhou Central Hospital. Written informed consents were obtained from all patients under protocols approved by the institutional review board. Normal Bone marrow CD34 cells were purchased from StemCell Technologies, Inc. CML and NBM CD34 cells were cultured using the same protocol as described in our previous studies [10]. Human K562 cells (ATCC, US) were cultured in RPMI1640 medium supplemented with 10% FBS, 4 mM L-glutamine and 50,000 units penicillin/streptomycin (Life Technologies, US). Ivermectin (ab141813, Abcam, US), Z-VADfmk (CalBiochem, US) and N-acetyl-L-cysteine (NAC, Sigma, US) were reconstituted in DMSO. 2.2. Flow cytometry 100, 000 cells were treated with drug alone or combination for 72 h in 24-well-plate. Cells were harvested for Annexin V-FITC and 7-AAD (BD Pharmingen, US) staining according to manufacture's instructions. Stained cells were analyzed by flow cytometry on a Beckman Coulter FC500. Annexin V-positive/7-AAD negative and Annexin V-positive/7-AAD negative were considered as early and late apoptosis, respectively. 2.3. Measurement of oxygen consumption rate (OCR) 37 C
OCR were measured at using an XF96 extracellular analyzer (Seahorse Bioscience). 10,000 cells were treated with drugs for 24 h and then transferred in XF96 well plate coated with BD Cell-Tak (BD Biosciences, MA, US). Cells were equilibrated to the un-buffered medium at 37 C in a CO2-free incubator for 1 h and transferred to XF96 extracellular analyzer. All injection reagents were adjusted to pH 7.4. OCR was measured under basal conditions, in the presence of the mitochondrial inhibitors oligomycin (0.5 mM) or antimycin A (0.25 mM), or in the presence of the mitochondrial uncoupler FCCP (0.2 mM) to assess maximal mitochondrial respiration. The Seahorse software calculated OCR automatically. 2.4. Determination of mitochondrial electron transport chain (ETC) complex I activities Cells were treated with drugs for 24 h. Mitochondrial ETC complex I activities were measured using Mitochondrial Complex Activity Assay Kits (Novagen, US) as determined by measuring the decrease in absorbance in mOD/min at room temperature and at 340 nm wavelength in kinetic mode (every min for 2 h) using a Spectramax M5 microplate reader. 2.5. Measurement of intracellular ROS and mitochondrial superoxide Cells were treated with drugs for 24 h. Cells were incubated with 10 mM CM-H2DCFDA and MitoSox Red (Life Technologies, US) for measuring intracellular ROS and mitochondrial superoxide, respectively. Absorbance at ex/em of 495/525 nm or ex/em of 510/ 580 were measured using a Spectramax M5 microplate reader.
2.7. Western blot analyses After 24 h drug treatment, cells were lysed by RIPA buffer (Life Technologies Inc, US). Equal amount of proteins were loaded and resolved using denaturing gel and analyzed by western blot using antibodies recognizing Akt, rS6, mOTR and phospho-Akt, rS6, mTOR and b-actin (Santa Cruz, US). 2.8. CML xenograft in SCID mouse All of the animal experiments were approved by the Institutional Animal Care and Use Committee of Yangtze University. 6week-old SCID mice (Institute of Laboratory Animals, Chinese Academy of Medical Sciences, China). K562 cells (10 106) were subcutaneously injected into mouse flank. When tumors reached ~200 mm3, the mice were treated with vehicle control (DMSO/Saline, 20%/80%), intraperitoneal doxorubicin at 0.5 mg/kg, ivermectin at various doses (detailed in figure legend) five days per week, or combination of doxorubicin and ivermectin. Tumor length and width were measured every two days and the volumes were calculated using the formula: length x width2 x 0.5236. 2.9. Statistical analyses The data are obtained from at least three independent experiments and expressed as mean and standard deviation (SD). Statistical analyses were performed by unpaired Student's t-test, with p-value < 0.05 considered statistically significant. 3. Results 3.1. Ivermectin selectively induces caspase-dependent apoptosis in CML cells and acts synergistically with BCR-ABL TKIs Successful eradication of cancer cells by nonsurgical means is approached via induction of apoptosis [11]. To demonstrate whether ivermectin can induce apoptosis in CML, we performed apoptosis assay using flow cytometry for Annexin V staining in CML cell line K562 as well as CD34 stem/progenitor cells derived from CML patients. We found that ivermectin at 5, 10 and 20 mM increased Annexin V percentage, demonstrating that ivermectin induces apoptosis in K562 and CML CD34 cells (Fig. 1A and B). Importantly, ivermectin at same concentration neither did not induce apoptosis nor induced significantly less apoptosis in NBM CD34 cells compared to CML CD34 cells (Fig. 1 B), suggesting that normal CD34 cells are less sensitive to ivermectin than CML CD34 cells. In addition, a pan-caspase inhibitor Z-VAD-fmk at 50 mM completely reversed the pro-apoptotic ability of ivermectin (Fig. 1D), demonstrating that ivermectin induces apoptosis in K562, CML and NBM CD34 cells through caspase-dependent pathway. We next investigated whether combination of ivermectin with BCR-ABL TKIs resulted in great efficacy than single drug alone in CML. We found that nilotinib at 0.1 mM, dasatinib at 50 nM and ivermectin at 5 mM induced ~30% apoptosis in K562 and CML CD34 cells (Fig. 2). However, when ivermectin (5 mM) is combined with nilotinib (0.1 mM) or dasatinib (50 nM), the combination
Please cite this article in press as: J. Wang, et al., Antibiotic ivermectin selectively induces apoptosis in chronic myeloid leukemia through inducing mitochondrial dysfunction and oxidative stress, Biochemical and Biophysical Research Communications (2018), https://doi.org/ 10.1016/j.bbrc.2018.02.063
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Fig. 1. Ivermectin selectively induces apoptosis in CML cells and acts synergistically with BCR-ABL TKIs. (A) Ivermectin dose-dependently induces apoptosis in K562 cells. (B) Ivermectin is more effective in inducing apoptosis in CML than NBM CD34 cells. Results are average of ten CML patients and healthy donor samples. (C) Addition of Z-VAD-fmk (50 mM) completely abolished the pro-apoptotic effect of ivermectin in CML and NBM cells. Combination of ivermectin with nilotinib or dasatinib induces significantly more apoptosis than ivermectin, nilotinib or dasatinib alone in K562 (D) and CML CD34 cells (E). Ivermectin at 5 mM, nilotinib at 0.1 mM and dasatinib at 50 nM were used in combination studies. *p < 0.05, compared to control, normal cells or single arm.
Fig. 2. Ivermectin impairs mitochondrial respiration in CML and NBM CD34 cells. Ivermectin at 10 and 20 mM decreases basal (A) and maximal (B) mitochondrial respiration and reduces mitochondrial respiratory complex I activity (C) in K562, CML and NBM CD34 cells. (D) Ivermectin decreases phosphorylation of Akt, mTOR and rS6 in K562 cells. *p < 0.05, compared to control.
Please cite this article in press as: J. Wang, et al., Antibiotic ivermectin selectively induces apoptosis in chronic myeloid leukemia through inducing mitochondrial dysfunction and oxidative stress, Biochemical and Biophysical Research Communications (2018), https://doi.org/ 10.1016/j.bbrc.2018.02.063
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induced ~95% apoptosis in K562 and CML CD34 cells (Fig. 1C and D). These data clearly demonstrate that combination of ivermectin with BCR-ABL TKIs is synergistic in killing CML cells.
3.2. Ivermectin impairs mitochondrial functions in CML and NBM CD34 cells Ivermectin has been reported to increase ROS levels which leads to leukemia cell death [9]. Given the fact that mitochondrial dysfunction is a major source of ROS [12], we investigated whether ivermectin disrupts mitochondrial functions in CML cells. We analyzed the mitochondrial respiration (indicated by OCR) and complex I activity in CML and NBM CD34 cells exposed to ivermectin. We found that NBM CD34 cells have less basal OCR level and reversed respiratory capacity than K562 or CML CD34 cells (Fig. 2A and B). It is noted that ivermectin at 5 mM only affected CML but not NBM CD34 cells (Fig. 2A and B). In addition, ivermectin at 10 and 20 mM significantly decreased basal and maximal mitochondrial respiration in K562, CML and NBM CD34 cells (Fig. 2A and B). In addition, ivermectin at 10 and 20 mM also inhibited mitochondrial respiratory complex I activity in CML and NBM CD34 cells (Fig. 2C). The molecular mechanisms of anti-cancer activities of ivermectin vary in different tumor types, including deactivation of PAK1 and KNP1, modulating P2X4 receptors and suppressing Akt/ mTOR [13e16]. We assessed the effects of ivermectin on Akt/ mTOR pathway as mTOR has been shown to control mitochondrial activity and biogenesis [17,18]. Our results show that ivermectin at
5, 10 and 20 mM significantly suppresses phosphorylation of Akt (Ser473), mTOR (S2481) and mTOR downstream molecule rS6 (S235/236) in K562 cells (Fig. 2D), suggesting that ivermectin inhibits Akt/mTOR pathway in CML. This result is also consistent with the Liu et al.'s work [16] that ivermectin targets tumor cells via inducing mitochondrial dysfunction and suppressing Akt/ mTOR.
3.3. Ivermectin selectively kills CML cells via inducing oxidative stress Superoxide is a precursor to many other forms of ROS and largely generated by mitochondrial electrons leakage due to mitochondrial dysfunction [19]. We next measured levels of mitochondrial superoxide and intracellular ROS levels in cells exposed to ivermectin. In line with ivermectin's ability in disrupting mitochondrial functions, we observed significantly increased levels of mitochondrial superoxide and intracellular ROS in K562 and CML CD34 cells in the presence of 5, 10 and 20 mM ivermectin (Fig. 3AeC). Ivermectin also increased 8-OHdG levels in K562 and CML CD34 cells (Fig. 3C). In addition, ivermectin increased levels of mitochondrial superoxide, intracellular ROS and 8-OHdG in normal T-cells, but to a significantly less extent than in K562 and CML CD34 cells (Fig. 3AeC). These data indicate that ivermectin selectively induces oxidative stress and causes oxidative damage in CML cells while NBM cells are largely unaffected. To further confirm the role of oxidative stress in the action of ivermectin in CML cells, we tested whether antioxidant NAC can rescue ivermectin's effects. 20 mM Ivermectin and 10 mM NAC were
Fig. 3. Ivermectin impairs mitochondrial function in CML and NBM cells. Ivermectin at 10 and 20 mM increases mitochondrial superoxide (A), intracellular ROS (B) and 8-OHdG (C) levels in CML and NBM cells. Ivermectin induces significantly less intracellular ROS (D), 8-OHdG (E) levels and apoptosis (F) in the presence of NAC in CML and NBM cells. *p < 0.05, compared to control or ivermectin alone.
Please cite this article in press as: J. Wang, et al., Antibiotic ivermectin selectively induces apoptosis in chronic myeloid leukemia through inducing mitochondrial dysfunction and oxidative stress, Biochemical and Biophysical Research Communications (2018), https://doi.org/ 10.1016/j.bbrc.2018.02.063
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concurrently added to the cells. We found that NAC rescued ivermectin-induced oxidative stress, oxidative damage and apoptosis in CML cells (Fig. 3DeF). In addition, the effects of ivermectin in NBM cells were also reversed by NAC (Fig. 3DeF). These data show that ivermectin selectively kills CML cells via inducing oxidative stress.
3.4. Ivermectin inhibits CML tumor growth in mice and significantly augments BCR-ABL TKI's inhibitory effect To confirm the inhibitory effects of ivermectin and investigate its translational potential, we established the CML xenograft mouse model by subcutaneous injection of K562 cells into the flank of SCID mice. We tested different concentrations of ivermectin and found that ivermectin at concentration below 5 mg/kg is not toxic to mice as no significant body weight loss and abnormal appearance were observed (Fig. 4A). In addition, ivermectin at 1, 2.5 and 5 mg/kg dose-dependently inhibited CML tumor growth in mice (Fig. 4B). More importantly, when combining ivermectin at 1 mg/kg with dasatinib at 0.5 mg/kg, the combination resulted in much greater efficacy than ivermectin or dasatinib alone (Fig. 4C). These clearly demonstrate the potent efficacy of ivermectin and synergism between ivermectin and BCR-ABL TKI in vivo. These results are also consistent with others on the anti-cancer activities of ivermectin in mouse model [15,16].
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4. Discussion BCR-ABL TKIs are effective to differentiated CML cells but not CD34 stem/progenitor cells which poses a therapeutic challenge in the management of this aggressive leukaemias [20]. Identification of novel agents that can sensitize CML CD34 cells to BCR-ABL TKIs may provide an alternative therapeutic strategy for CML treatment. Mitochondrial metabolism has been a promising target particularly in blood cancer because malignant hematopoietic cells reply more on mitochondrial functions compared to normal counterparts [21e23]. Ivermectin is an anthelminthic drug and has been recently identified as a novel type of anti-cancer drug [7e9,24]. In this work, we demonstrated the efficacy of ivermectin on CML CD34 cells. Our work suggests that ivermectin is a promising candidate for CML treatment as it selectively induces apoptosis of CML CD34 cells while NBM CD34 cells are largely unaffected. In addition, we identified mitochondria as the target of ivermectin in CML. We tested the efficacy of ivermectin in not only CML cell line but also primary CD34 cells derived from patients with CML. The results show that ivermectin induces caspase-dependent apoptosis in K562 and primary CML CD34 cells in a similar manner (Fig. 1AeC). Ivermectin has been reported to induce a mixed apoptotic and necrotic mode of breast cancer cell death [13]. However, the complete rather than partial rescue on the effects of ivermectin by a pan caspase-inhibitor Z-VAD-fmk demonstrates that ivermectin induces CML cell death via caspase-dependent apoptosis (Fig. 1C).
Fig. 4. Ivermectin significantly inhibits CML growth in xenograft mouse model and acts synergistically with doxorubicin. (A) Ivermectin up to 5 mg/kg does not affect mouse body weight. (B) Ivermectin inhibits K562 growth in xenograft mouse model in a dose-dependent manner. (C) Combination of ivermectin with dasatinib achieves significantly greater efficacy in inhibiting K562 growth than ivermectin or dasatinib alone. Ivermectin at 2.5 mg/kg and dasatinib at 1 mg/kg were used for in combination studies. *p < 0.05, compared to control or single arm.
Please cite this article in press as: J. Wang, et al., Antibiotic ivermectin selectively induces apoptosis in chronic myeloid leukemia through inducing mitochondrial dysfunction and oxidative stress, Biochemical and Biophysical Research Communications (2018), https://doi.org/ 10.1016/j.bbrc.2018.02.063
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Interestingly, ivermectin also significantly sensitizes K562 and primary CML CD34 cells to BCR-ABL TKIs, such as nilotinib and dasatinib (Fig. 1D and E). The data obtained on primary cells is a direct evidence to indicate the efficacy of ivermectin on CML patients. In addition, the in vivo efficacy of ivermectin has also been demonstrated in CML xenograft mouse model (Fig. 4). The synergy observed between ivermectin and BCR-ABL TKIs in CML are consistent with the previous work that ivermectin synergizes with cytarabine and daunorubicin in acute myeloid leukemia [9]. The molecular mechanisms of ivermectin's action in cancer vary in different types of cancer. Ivermectin has been demonstrated to exert anti-cancer activities via blocking the PAK/Akt axis, WNT-TCF pathway in solid cancer [6e8] and inducing chloride-dependent membrane hyperpolarization in blood cancer [9]. We show that ivermectin inhibits mitochondrial respiration via decreasing mitochondrial complex I activity, leading to energy crisis, oxidative stress and damage in CML cells (Fig. 2). 8-OHdG is often used as a marker of oxidative DNA damage. It seems that the generation of 8OHdG by ivermectin is the consequence of oxidative stress which is caused by mitochondrial dysfunction. This is also evidenced by the findings that NBM CD34 cells have less amount of 8-OHdG as they are less sensitive to mitochondrial dysfunction compared to CML CD34 cells (Fig. 3C). The potential genetic toxicity of 8-OHdG in cells should be considered during the development of ivermectin or mitochondria-targeted drugs. An antioxidant NAC significantly abolishes the effects of ivermectin in inducing oxidative stress and apoptosis (Fig. 3DeF), further confirming that mitochondrial dysfunction and oxidative stress are required for ivermectin's action in CML. Importantly, apart from anti-CML activities, we are the first to show the selectivity of ivermectin between hematopoietic malignant and normal cells as ivermectin is significantly more effective in inducing apoptosis in CML CD34 cells compared to their normal counterparts (Fig. 1). Both lymphoma T cells and AML CD34 cells have been reported to have higher mitochondrial biogenesis and depends more on mitochondrial respiration than their normal counterparts [22,25]. Therefore, targeting mitochondrial respiration by pharmacological inhibitors (eg, pyrvinium and tigecycline) or genetic approach (eg, depletion of EF-Tu) selectively inhibits AML and lymphoma cells while sparing normal cells [22,25,26]. We would like to highlight that the mechanism of the action of ivermectin on CML is mitochondrial respiration inhibition. This explains the different sensitivity of CML and NBM CD34 cells to ivermectin. Targeting mitochondria has become a promising therapeutic strategy in blood cancer because hematopoietic malignant cells depend more on mitochondrial respiration compared to normal counterparts [23,27,28]. In agreement with the previous work [25], we also observe the higher levels of mitochondrial respiration in CML than NBM CD34 cells (Fig. 2A and B). Although ivermectin at 10 and 20 mM inhibits mitochondrial respiration in NBM CD34 cells in a similar manner as in CML CD34 cells (Fig. 2A and B), ivermectin induces significantly less apoptosis in NBM than CML CD34 cells (Fig. 1). These suggest that CML CD34 cells are more sensitive than NBM CD34 cells to mitochondrial respiration inhibition. This is further confirmed by our findings that ivermectin at 10 and 20 mM induces less oxidative stress and damage (Fig. 3). Moreover, Akt/ mTOR pathway inhibition by ivermectin (Fig. 2D) suggests the association between mitochondrial dysfunctions and Akt/mTOR oncogenic signaling pathways. In conclusion, our work is the first to demonstrate that ivermectin exerts selective toxicity and enhances BCR-ABL TKI's effects in CML CD34 cells via inducing mitochondrial dysfunctions and oxidative stress. Our work suggests that ivermectin is a useful addition to the treatment armamentarium for CML.
Conflicts of interest All authors declare no conflict of interest. Acknowledgement We acknowledge the many helpful comments and discussions with our colleagues. This work was supported by research grants provided by Jingzhou Central Hospital (2013061816) and Yangtze University (2015610185). Transparency document Transparency document related to this article can be found online at https://doi.org/10.1016/j.bbrc.2018.02.063. References [1] C.L. Sawyers, Chronic myeloid leukemia, N. Engl. J. Med. 340 (1999) 1330e1340. [2] T. Holyoake, X.Y. Jiang, C. Eaves, A. Eaves, Isolation of a highly quiescent subpopulation of primitive leukemic cells in chronic myeloid leukemia, Blood 94 (1999) 2056e2064. [3] R. Hehlmann, How I treat CML blast crisis, Blood 120 (2012) 737e747. [4] C.H. Jamieson, L.E. Ailles, S.J. Dylla, M. Muijtjens, C. Jones, J.L. Zehnder, J. Gotlib, K. Li, M.G. Manz, A. Keating, C.L. Sawyers, I.L. Weissman, Granulocytemacrophage progenitors as candidate leukemic stem cells in blast-crisis CML, N. Engl. J. Med. 351 (2004) 657e667. [5] W.A. Stolk, M. Walker, L.E. Coffeng, M.G. Basanez, S.J. de Vlas, Required duration of mass ivermectin treatment for onchocerciasis elimination in Africa: a comparative modelling analysis, Parasites Vectors 8 (2015) 552. [6] K. Wang, W. Gao, Q. Dou, H. Chen, Q. Li, E.C. Nice, C. Huang, Ivermectin induces PAK1-mediated cytostatic autophagy in breast cancer, Autophagy 12 (2016) 2498e2499. [7] Q. Dou, H.N. Chen, K. Wang, K. Yuan, Y. Lei, K. Li, J. Lan, Y. Chen, Z. Huang, N. Xie, L. Zhang, R. Xiang, E.C. Nice, Y. Wei, C. Huang, Ivermectin induces cytostatic autophagy by blocking the PAK1/Akt Axis in breast cancer, Canc. Res. 76 (2016) 4457e4469. [8] A. Melotti, C. Mas, M. Kuciak, A. Lorente-Trigos, I. Borges, A. Ruiz i Altaba, The river blindness drug Ivermectin and related macrocyclic lactones inhibit WNT-TCF pathway responses in human cancer, EMBO Mol. Med. 6 (2014) 1263e1278. [9] S. Sharmeen, M. Skrtic, M.A. Sukhai, R. Hurren, M. Gronda, X. Wang, S.B. Fonseca, H. Sun, T.E. Wood, R. Ward, M.D. Minden, R.A. Batey, A. Datti, J. Wrana, S.O. Kelley, A.D. Schimmer, The antiparasitic agent ivermectin induces chloride-dependent membrane hyperpolarization and cell death in leukemia cells, Blood 116 (2010) 3593e3603. [10] J. Wang, J. Hu, Z. Jin, H. Wan, The sensitivity of chronic myeloid leukemia CD34 cells to Bcr-Abl tyrosine kinase inhibitors is modulated by ceramide levels, Leuk. Res. 47 (2016) 32e40. [11] M. Hassan, H. Watari, A. AbuAlmaaty, Y. Ohba, N. Sakuragi, Apoptosis and molecular targeting therapy in cancer, BioMed Res. Int. 2014 (2014) 150845. [12] E.J. Lesnefsky, S. Moghaddas, B. Tandler, J. Kerner, C.L. Hoppel, Mitochondrial dysfunction in cardiac disease: ischemiaereperfusion, aging, and heart failure, J. Mol. Cell. Cardiol. 33 (2001) 1065e1089. [13] D. Draganov, S. Gopalakrishna-Pillai, Y.R. Chen, N. Zuckerman, S. Moeller, C. Wang, D. Ann, P.P. Lee, Modulation of P2X4/P2X7/Pannexin-1 sensitivity to extracellular ATP via Ivermectin induces a non-apoptotic and inflammatory form of cancer cell death, Sci. Rep. 5 (2015) 16222. [14] H. Hashimoto, T. Sudo, H. Maruta, R. Nishimura, The direct PAK1 inhibitor, TAT-PAK18, blocks preferentially the growth of human ovarian cancer cell lines in which PAK1 is abnormally activated by autophosphorylation at Thr 423, Drug Discov. Ther. 4 (2010) 1e4. [15] M. Kodama, T. Kodama, J.Y. Newberg, H. Katayama, M. Kobayashi, S.M. Hanash, K. Yoshihara, Z. Wei, J.C. Tien, R. Rangel, K. Hashimoto, S. Mabuchi, K. Sawada, T. Kimura, N.G. Copeland, N.A. Jenkins, In vivo loss-offunction screens identify KPNB1 as a new druggable oncogene in epithelial ovarian cancer, Proc. Natl. Acad. Sci. U. S. A. 114 (2017) E7301eE7310. [16] Y. Liu, S. Fang, Q. Sun, B. Liu, Anthelmintic drug ivermectin inhibits angiogenesis, growth and survival of glioblastoma through inducing mitochondrial dysfunction and oxidative stress, Biochem. Biophys. Res. Commun. 480 (2016) 415e421. [17] M. Morita, S.P. Gravel, V. Chenard, K. Sikstrom, L. Zheng, T. Alain, V. Gandin, D. Avizonis, M. Arguello, C. Zakaria, S. McLaughlan, Y. Nouet, A. Pause, M. Pollak, E. Gottlieb, O. Larsson, J. St-Pierre, I. Topisirovic, N. Sonenberg, mTORC1 controls mitochondrial activity and biogenesis through 4E-BPdependent translational regulation, Cell Metab. 18 (2013) 698e711. [18] A. Ramanathan, S.L. Schreiber, Direct control of mitochondrial function by mTOR, Proc. Natl. Acad. Sci. U. S. A. 106 (2009) 22229e22232.
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Antibiotic ivermectin selectively induces apoptosis in chronic myeloid leukemia through inducing mitochondrial dysfunction and oxidative stress Jiaqiao Wang, Yanhua Xu, Huihui Wan, June Hu* Department of Oncology, Jingzhou Central Hospital, The Second Clinical Medical College, Yangtze University, Jing Zhou, People’s Republic of China
a r t i c l e i n f o
a b s t r a c t
Article history: Received 5 February 2018 Accepted 7 February 2018 Available online xxx
Mitochondria has been a promising target in blood cancer given their unique dependencies on mitochondrial functions compared to normal hematopoietic cells. In line with this concept, we show that an anthelminthic drug ivermectin selectively kills chronic myeloid leukemia (CML) cells via inducing mitochondrial dysfunctions and oxidative stress. Ivermectin is significantly more effective in inducing caspase-dependent apoptosis in CML cell line K562 and primary CML CD34 than normal bone marrow (NBM) CD34 cells. Ivermectin also augments in vitro and in vivo efficacy of standard CML tyrosine kinase inhibitors. Mechanistically, ivermectin inhibits respiratory complex I activity and suppresses mitochondrial respiration in K562 and CML CD34 cells. Interestingly, we demonstrate that mitochondrial respiration are lower in NBM CD34 compared to malignant CD34 cells. In addition, ivermectin also induces mitochondrial dysfunctions in NBM CD34 cells in a similar manner as in CML CD34 cells whereas NBM CD34 cells are significantly less sensitive to ivermectin than CML CD34 cells. These suggest that NBM CD34 cells are more tolerable to mitochondrial dysfunctions than CML CD34 cells. Consistently, ivermectin induces higher levels of oxidative stress and damage in CML than normal counterparts. Antioxidant NAC rescues ivermectin's effects, confirming oxidative stress as the mechanism of its action in CML. Our work provides the fundamental evidence to repurpose ivermectin for CML treatment. Our work also highlights the therapeutic value of targeting mitochondria respiration in CML. © 2018 Elsevier Inc. All rights reserved.
Keywords: Ivermectin CML Mitochondrial respiration Oxidative damage Selectivity
1. Introduction Chronic myeloid leukemia (CML) is a hematological stem cell malignancy and characterized by the presence of Philadelphia chromosome that generates the BCR-ABL oncogene [1]. BCR-ABLpositive hematopoietic CD34 stem/progenitor cells have a proliferative advantage which eventually displace residual normal hematopoiesis [2]. Treatment of BCR-ABL tyrosine kinase inhibitors (TKIs), such as nilotinib and dasatinib, has significantly improved clinical outcomes. However, primitive leukemia CD34 stem/progenitor cells are retained even in patients achieving remission, suggesting that treatment with TKIs alone is insufficient to cure CML [3,4].
* Corresponding author. Department of Oncology, Jingzhou Central Hospital, The Second Clinical Medical College, Yangtze University, Renmin Road 1, 434020 Jing Zhou, People’s Republic of China. E-mail address: [email protected] (J. Hu).
Ivermectin is an anthelminthic drug used for the treatment of many types of parasites with unknown mechanism of action [5]. However, ivermectin has been recently identified to exhibit potent anti-cancer activities. It induces cytostatic autophagy and necrosis in breast cancer cells via blocking the PAK/Akt axis and modulating P2X4/P2X7 signaling, respectively [6,7]. Ivermectin inhibits xenograft growth of many types of solid cancer without obvious side effects via inhibiting WNT-TCF pathway [8]. It also induces chloride-dependent membrane hyperpolarization and cell death in blood cancer, such as leukemia [9]. In this study, we investigated the effect and selectivity of ivermectin in CML cell line K562, primary CML CD34 and normal bone marrow (NBM) CD34 cells. We next investigated whether ivermectin can sensitize CML cells to multiple BCR-ABL TKIs. Finally, we determined the target of ivermectin in CML cells. We are the first to demonstrate that ivermectin induces apoptosis in CML cells while sparing normal hematopoietic cells, via disrupting mitochondrial functions and inducing oxidative stress. The different dependencies of CML and normal CD34 cells to mitochondrial functions might
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contribute to their sensitivities to ivermectin treatment.
2.6. Oxidative DNA damage
2. Materials and methods
Cells were treated with drug or combination for 24 h. Cells were harvested and DNA was extracted using the DNEasy Mini Kit (Qiagen). 8-OHdG levels were quantified using the OxiSelect Oxidative DNA Damage ELISA Kit (Cell Biolabs) according to manufacture's protocol. Absorbance was read on a Spectramax M5 Microplate reader at 450 nm.
2.1. Primary cells, cell culture and drug reconstitution Human peripheral blood mononuclear cells were isolated using Ficoll separation. Primary CML CD34 cells were further purified using CD34 MicroBead isolation kit (Miltenyi Biotec, Germany) according to manufacturer's instructions. CML samples were obtained from patients seen at Jingzhou Central Hospital. Written informed consents were obtained from all patients under protocols approved by the institutional review board. Normal Bone marrow CD34 cells were purchased from StemCell Technologies, Inc. CML and NBM CD34 cells were cultured using the same protocol as described in our previous studies [10]. Human K562 cells (ATCC, US) were cultured in RPMI1640 medium supplemented with 10% FBS, 4 mM L-glutamine and 50,000 units penicillin/streptomycin (Life Technologies, US). Ivermectin (ab141813, Abcam, US), Z-VADfmk (CalBiochem, US) and N-acetyl-L-cysteine (NAC, Sigma, US) were reconstituted in DMSO. 2.2. Flow cytometry 100, 000 cells were treated with drug alone or combination for 72 h in 24-well-plate. Cells were harvested for Annexin V-FITC and 7-AAD (BD Pharmingen, US) staining according to manufacture's instructions. Stained cells were analyzed by flow cytometry on a Beckman Coulter FC500. Annexin V-positive/7-AAD negative and Annexin V-positive/7-AAD negative were considered as early and late apoptosis, respectively. 2.3. Measurement of oxygen consumption rate (OCR) 37 C
OCR were measured at using an XF96 extracellular analyzer (Seahorse Bioscience). 10,000 cells were treated with drugs for 24 h and then transferred in XF96 well plate coated with BD Cell-Tak (BD Biosciences, MA, US). Cells were equilibrated to the un-buffered medium at 37 C in a CO2-free incubator for 1 h and transferred to XF96 extracellular analyzer. All injection reagents were adjusted to pH 7.4. OCR was measured under basal conditions, in the presence of the mitochondrial inhibitors oligomycin (0.5 mM) or antimycin A (0.25 mM), or in the presence of the mitochondrial uncoupler FCCP (0.2 mM) to assess maximal mitochondrial respiration. The Seahorse software calculated OCR automatically. 2.4. Determination of mitochondrial electron transport chain (ETC) complex I activities Cells were treated with drugs for 24 h. Mitochondrial ETC complex I activities were measured using Mitochondrial Complex Activity Assay Kits (Novagen, US) as determined by measuring the decrease in absorbance in mOD/min at room temperature and at 340 nm wavelength in kinetic mode (every min for 2 h) using a Spectramax M5 microplate reader. 2.5. Measurement of intracellular ROS and mitochondrial superoxide Cells were treated with drugs for 24 h. Cells were incubated with 10 mM CM-H2DCFDA and MitoSox Red (Life Technologies, US) for measuring intracellular ROS and mitochondrial superoxide, respectively. Absorbance at ex/em of 495/525 nm or ex/em of 510/ 580 were measured using a Spectramax M5 microplate reader.
2.7. Western blot analyses After 24 h drug treatment, cells were lysed by RIPA buffer (Life Technologies Inc, US). Equal amount of proteins were loaded and resolved using denaturing gel and analyzed by western blot using antibodies recognizing Akt, rS6, mOTR and phospho-Akt, rS6, mTOR and b-actin (Santa Cruz, US). 2.8. CML xenograft in SCID mouse All of the animal experiments were approved by the Institutional Animal Care and Use Committee of Yangtze University. 6week-old SCID mice (Institute of Laboratory Animals, Chinese Academy of Medical Sciences, China). K562 cells (10 106) were subcutaneously injected into mouse flank. When tumors reached ~200 mm3, the mice were treated with vehicle control (DMSO/Saline, 20%/80%), intraperitoneal doxorubicin at 0.5 mg/kg, ivermectin at various doses (detailed in figure legend) five days per week, or combination of doxorubicin and ivermectin. Tumor length and width were measured every two days and the volumes were calculated using the formula: length x width2 x 0.5236. 2.9. Statistical analyses The data are obtained from at least three independent experiments and expressed as mean and standard deviation (SD). Statistical analyses were performed by unpaired Student's t-test, with p-value < 0.05 considered statistically significant. 3. Results 3.1. Ivermectin selectively induces caspase-dependent apoptosis in CML cells and acts synergistically with BCR-ABL TKIs Successful eradication of cancer cells by nonsurgical means is approached via induction of apoptosis [11]. To demonstrate whether ivermectin can induce apoptosis in CML, we performed apoptosis assay using flow cytometry for Annexin V staining in CML cell line K562 as well as CD34 stem/progenitor cells derived from CML patients. We found that ivermectin at 5, 10 and 20 mM increased Annexin V percentage, demonstrating that ivermectin induces apoptosis in K562 and CML CD34 cells (Fig. 1A and B). Importantly, ivermectin at same concentration neither did not induce apoptosis nor induced significantly less apoptosis in NBM CD34 cells compared to CML CD34 cells (Fig. 1 B), suggesting that normal CD34 cells are less sensitive to ivermectin than CML CD34 cells. In addition, a pan-caspase inhibitor Z-VAD-fmk at 50 mM completely reversed the pro-apoptotic ability of ivermectin (Fig. 1D), demonstrating that ivermectin induces apoptosis in K562, CML and NBM CD34 cells through caspase-dependent pathway. We next investigated whether combination of ivermectin with BCR-ABL TKIs resulted in great efficacy than single drug alone in CML. We found that nilotinib at 0.1 mM, dasatinib at 50 nM and ivermectin at 5 mM induced ~30% apoptosis in K562 and CML CD34 cells (Fig. 2). However, when ivermectin (5 mM) is combined with nilotinib (0.1 mM) or dasatinib (50 nM), the combination
Please cite this article in press as: J. Wang, et al., Antibiotic ivermectin selectively induces apoptosis in chronic myeloid leukemia through inducing mitochondrial dysfunction and oxidative stress, Biochemical and Biophysical Research Communications (2018), https://doi.org/ 10.1016/j.bbrc.2018.02.063
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Fig. 1. Ivermectin selectively induces apoptosis in CML cells and acts synergistically with BCR-ABL TKIs. (A) Ivermectin dose-dependently induces apoptosis in K562 cells. (B) Ivermectin is more effective in inducing apoptosis in CML than NBM CD34 cells. Results are average of ten CML patients and healthy donor samples. (C) Addition of Z-VAD-fmk (50 mM) completely abolished the pro-apoptotic effect of ivermectin in CML and NBM cells. Combination of ivermectin with nilotinib or dasatinib induces significantly more apoptosis than ivermectin, nilotinib or dasatinib alone in K562 (D) and CML CD34 cells (E). Ivermectin at 5 mM, nilotinib at 0.1 mM and dasatinib at 50 nM were used in combination studies. *p < 0.05, compared to control, normal cells or single arm.
Fig. 2. Ivermectin impairs mitochondrial respiration in CML and NBM CD34 cells. Ivermectin at 10 and 20 mM decreases basal (A) and maximal (B) mitochondrial respiration and reduces mitochondrial respiratory complex I activity (C) in K562, CML and NBM CD34 cells. (D) Ivermectin decreases phosphorylation of Akt, mTOR and rS6 in K562 cells. *p < 0.05, compared to control.
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induced ~95% apoptosis in K562 and CML CD34 cells (Fig. 1C and D). These data clearly demonstrate that combination of ivermectin with BCR-ABL TKIs is synergistic in killing CML cells.
3.2. Ivermectin impairs mitochondrial functions in CML and NBM CD34 cells Ivermectin has been reported to increase ROS levels which leads to leukemia cell death [9]. Given the fact that mitochondrial dysfunction is a major source of ROS [12], we investigated whether ivermectin disrupts mitochondrial functions in CML cells. We analyzed the mitochondrial respiration (indicated by OCR) and complex I activity in CML and NBM CD34 cells exposed to ivermectin. We found that NBM CD34 cells have less basal OCR level and reversed respiratory capacity than K562 or CML CD34 cells (Fig. 2A and B). It is noted that ivermectin at 5 mM only affected CML but not NBM CD34 cells (Fig. 2A and B). In addition, ivermectin at 10 and 20 mM significantly decreased basal and maximal mitochondrial respiration in K562, CML and NBM CD34 cells (Fig. 2A and B). In addition, ivermectin at 10 and 20 mM also inhibited mitochondrial respiratory complex I activity in CML and NBM CD34 cells (Fig. 2C). The molecular mechanisms of anti-cancer activities of ivermectin vary in different tumor types, including deactivation of PAK1 and KNP1, modulating P2X4 receptors and suppressing Akt/ mTOR [13e16]. We assessed the effects of ivermectin on Akt/ mTOR pathway as mTOR has been shown to control mitochondrial activity and biogenesis [17,18]. Our results show that ivermectin at
5, 10 and 20 mM significantly suppresses phosphorylation of Akt (Ser473), mTOR (S2481) and mTOR downstream molecule rS6 (S235/236) in K562 cells (Fig. 2D), suggesting that ivermectin inhibits Akt/mTOR pathway in CML. This result is also consistent with the Liu et al.'s work [16] that ivermectin targets tumor cells via inducing mitochondrial dysfunction and suppressing Akt/ mTOR.
3.3. Ivermectin selectively kills CML cells via inducing oxidative stress Superoxide is a precursor to many other forms of ROS and largely generated by mitochondrial electrons leakage due to mitochondrial dysfunction [19]. We next measured levels of mitochondrial superoxide and intracellular ROS levels in cells exposed to ivermectin. In line with ivermectin's ability in disrupting mitochondrial functions, we observed significantly increased levels of mitochondrial superoxide and intracellular ROS in K562 and CML CD34 cells in the presence of 5, 10 and 20 mM ivermectin (Fig. 3AeC). Ivermectin also increased 8-OHdG levels in K562 and CML CD34 cells (Fig. 3C). In addition, ivermectin increased levels of mitochondrial superoxide, intracellular ROS and 8-OHdG in normal T-cells, but to a significantly less extent than in K562 and CML CD34 cells (Fig. 3AeC). These data indicate that ivermectin selectively induces oxidative stress and causes oxidative damage in CML cells while NBM cells are largely unaffected. To further confirm the role of oxidative stress in the action of ivermectin in CML cells, we tested whether antioxidant NAC can rescue ivermectin's effects. 20 mM Ivermectin and 10 mM NAC were
Fig. 3. Ivermectin impairs mitochondrial function in CML and NBM cells. Ivermectin at 10 and 20 mM increases mitochondrial superoxide (A), intracellular ROS (B) and 8-OHdG (C) levels in CML and NBM cells. Ivermectin induces significantly less intracellular ROS (D), 8-OHdG (E) levels and apoptosis (F) in the presence of NAC in CML and NBM cells. *p < 0.05, compared to control or ivermectin alone.
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concurrently added to the cells. We found that NAC rescued ivermectin-induced oxidative stress, oxidative damage and apoptosis in CML cells (Fig. 3DeF). In addition, the effects of ivermectin in NBM cells were also reversed by NAC (Fig. 3DeF). These data show that ivermectin selectively kills CML cells via inducing oxidative stress.
3.4. Ivermectin inhibits CML tumor growth in mice and significantly augments BCR-ABL TKI's inhibitory effect To confirm the inhibitory effects of ivermectin and investigate its translational potential, we established the CML xenograft mouse model by subcutaneous injection of K562 cells into the flank of SCID mice. We tested different concentrations of ivermectin and found that ivermectin at concentration below 5 mg/kg is not toxic to mice as no significant body weight loss and abnormal appearance were observed (Fig. 4A). In addition, ivermectin at 1, 2.5 and 5 mg/kg dose-dependently inhibited CML tumor growth in mice (Fig. 4B). More importantly, when combining ivermectin at 1 mg/kg with dasatinib at 0.5 mg/kg, the combination resulted in much greater efficacy than ivermectin or dasatinib alone (Fig. 4C). These clearly demonstrate the potent efficacy of ivermectin and synergism between ivermectin and BCR-ABL TKI in vivo. These results are also consistent with others on the anti-cancer activities of ivermectin in mouse model [15,16].
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4. Discussion BCR-ABL TKIs are effective to differentiated CML cells but not CD34 stem/progenitor cells which poses a therapeutic challenge in the management of this aggressive leukaemias [20]. Identification of novel agents that can sensitize CML CD34 cells to BCR-ABL TKIs may provide an alternative therapeutic strategy for CML treatment. Mitochondrial metabolism has been a promising target particularly in blood cancer because malignant hematopoietic cells reply more on mitochondrial functions compared to normal counterparts [21e23]. Ivermectin is an anthelminthic drug and has been recently identified as a novel type of anti-cancer drug [7e9,24]. In this work, we demonstrated the efficacy of ivermectin on CML CD34 cells. Our work suggests that ivermectin is a promising candidate for CML treatment as it selectively induces apoptosis of CML CD34 cells while NBM CD34 cells are largely unaffected. In addition, we identified mitochondria as the target of ivermectin in CML. We tested the efficacy of ivermectin in not only CML cell line but also primary CD34 cells derived from patients with CML. The results show that ivermectin induces caspase-dependent apoptosis in K562 and primary CML CD34 cells in a similar manner (Fig. 1AeC). Ivermectin has been reported to induce a mixed apoptotic and necrotic mode of breast cancer cell death [13]. However, the complete rather than partial rescue on the effects of ivermectin by a pan caspase-inhibitor Z-VAD-fmk demonstrates that ivermectin induces CML cell death via caspase-dependent apoptosis (Fig. 1C).
Fig. 4. Ivermectin significantly inhibits CML growth in xenograft mouse model and acts synergistically with doxorubicin. (A) Ivermectin up to 5 mg/kg does not affect mouse body weight. (B) Ivermectin inhibits K562 growth in xenograft mouse model in a dose-dependent manner. (C) Combination of ivermectin with dasatinib achieves significantly greater efficacy in inhibiting K562 growth than ivermectin or dasatinib alone. Ivermectin at 2.5 mg/kg and dasatinib at 1 mg/kg were used for in combination studies. *p < 0.05, compared to control or single arm.
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Interestingly, ivermectin also significantly sensitizes K562 and primary CML CD34 cells to BCR-ABL TKIs, such as nilotinib and dasatinib (Fig. 1D and E). The data obtained on primary cells is a direct evidence to indicate the efficacy of ivermectin on CML patients. In addition, the in vivo efficacy of ivermectin has also been demonstrated in CML xenograft mouse model (Fig. 4). The synergy observed between ivermectin and BCR-ABL TKIs in CML are consistent with the previous work that ivermectin synergizes with cytarabine and daunorubicin in acute myeloid leukemia [9]. The molecular mechanisms of ivermectin's action in cancer vary in different types of cancer. Ivermectin has been demonstrated to exert anti-cancer activities via blocking the PAK/Akt axis, WNT-TCF pathway in solid cancer [6e8] and inducing chloride-dependent membrane hyperpolarization in blood cancer [9]. We show that ivermectin inhibits mitochondrial respiration via decreasing mitochondrial complex I activity, leading to energy crisis, oxidative stress and damage in CML cells (Fig. 2). 8-OHdG is often used as a marker of oxidative DNA damage. It seems that the generation of 8OHdG by ivermectin is the consequence of oxidative stress which is caused by mitochondrial dysfunction. This is also evidenced by the findings that NBM CD34 cells have less amount of 8-OHdG as they are less sensitive to mitochondrial dysfunction compared to CML CD34 cells (Fig. 3C). The potential genetic toxicity of 8-OHdG in cells should be considered during the development of ivermectin or mitochondria-targeted drugs. An antioxidant NAC significantly abolishes the effects of ivermectin in inducing oxidative stress and apoptosis (Fig. 3DeF), further confirming that mitochondrial dysfunction and oxidative stress are required for ivermectin's action in CML. Importantly, apart from anti-CML activities, we are the first to show the selectivity of ivermectin between hematopoietic malignant and normal cells as ivermectin is significantly more effective in inducing apoptosis in CML CD34 cells compared to their normal counterparts (Fig. 1). Both lymphoma T cells and AML CD34 cells have been reported to have higher mitochondrial biogenesis and depends more on mitochondrial respiration than their normal counterparts [22,25]. Therefore, targeting mitochondrial respiration by pharmacological inhibitors (eg, pyrvinium and tigecycline) or genetic approach (eg, depletion of EF-Tu) selectively inhibits AML and lymphoma cells while sparing normal cells [22,25,26]. We would like to highlight that the mechanism of the action of ivermectin on CML is mitochondrial respiration inhibition. This explains the different sensitivity of CML and NBM CD34 cells to ivermectin. Targeting mitochondria has become a promising therapeutic strategy in blood cancer because hematopoietic malignant cells depend more on mitochondrial respiration compared to normal counterparts [23,27,28]. In agreement with the previous work [25], we also observe the higher levels of mitochondrial respiration in CML than NBM CD34 cells (Fig. 2A and B). Although ivermectin at 10 and 20 mM inhibits mitochondrial respiration in NBM CD34 cells in a similar manner as in CML CD34 cells (Fig. 2A and B), ivermectin induces significantly less apoptosis in NBM than CML CD34 cells (Fig. 1). These suggest that CML CD34 cells are more sensitive than NBM CD34 cells to mitochondrial respiration inhibition. This is further confirmed by our findings that ivermectin at 10 and 20 mM induces less oxidative stress and damage (Fig. 3). Moreover, Akt/ mTOR pathway inhibition by ivermectin (Fig. 2D) suggests the association between mitochondrial dysfunctions and Akt/mTOR oncogenic signaling pathways. In conclusion, our work is the first to demonstrate that ivermectin exerts selective toxicity and enhances BCR-ABL TKI's effects in CML CD34 cells via inducing mitochondrial dysfunctions and oxidative stress. Our work suggests that ivermectin is a useful addition to the treatment armamentarium for CML.
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Please cite this article in press as: J. Wang, et al., Antibiotic ivermectin selectively induces apoptosis in chronic myeloid leukemia through inducing mitochondrial dysfunction and oxidative stress, Biochemical and Biophysical Research Communications (2018), https://doi.org/ 10.1016/j.bbrc.2018.02.063