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β-catenin inhibition
Biomedicine & Pharmacotherapy 107 (2018) 1294–1301
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Volatile anesthetics sevoflurane targets leukemia stem/progenitor cells via Wnt/β-catenin inhibition
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Xuguang Ruana, Weihang Jianga, Pingrui Chenga, Lingyan Huanga, Xuelan Lia, Yingyi Hea, ⁎ Minyi Maia, Zhimin Tanb, a b
Department of Anesthesiology, Panyu Central Hospital, Guangzhou, China Department of Anesthesiology, Shenzhen Hospital, Southern Medical University, Xinhu Road 1333, Bao’an District, Shenzhen, China
A R T I C LE I N FO
A B S T R A C T
Keywords: Sevoflurane Volatile anesthetics Wnt/β-catenin Stem/progenitors Leukemia
Most of the studies regarding the direct effect of anesthetics on tumour cells are focused on opioids and voltagegated sodium channels. Little is known on the effect of volatile anesthetics on tumour progression. In this study, we show that sevoflurane, a volatile anesthetic, negatively affects chronic myeloid leukemia (CML) CD34 stem/ progenitor cells’ biological properties. Sevoflurane significantly inhibits the growth of a panel of CML cell lines in a dose-dependent manner without affecting their survival. It also inhibits proliferation, differentiation and selfrenewal capacities but not survival of CML CD34 cells. In addition, sevoflurane significantly augments dasatinib’s efficacy in CML cell lines and stem/progenitors. Mechanistically, sevoflurane dose-dependently decreases levels of β-catenin and c-Myc but not phospho-P38 MAPK in K562 and CML CD34 cells. The decreased Wnt/ βcatenin activity and the reduced levels of Wnt/β-catenin-targeted transcriptions are observed in CML cells exposed to sevoflurane. The complete rescue of the inhibitory effects of sevoflurane in K562 and CML CD34 cells by β-catenin stabilization using both genetic and pharmacological approaches further demonstrates that sevoflurane acts on CML cells via a β-catenin-dependent manner. Our results clearly show the direct and negative effects of sevoflurane on the leukemia cell lines as well as leukemia stem/progenitors. Our findings also reveal Wnt/β-catenin as the target of volatile anesthetics.
1. Introduction A number of retrospective studies suggest that the type of anesthetics as well as anesthetic techniques chosen for cancer patients during surgery or chronic pain control management have potential influence on the long-term outcome of the disease [1]. For example, local anaesthesia is associated with reduced cancer recurrence and improved overall survival [2]. Studies using pre-clinical models demonstrate that local anaesthesia including ropivacaine, lidocaine and bupivacaine, inhibit various tumour growth and metastasis, and induce tumour cell death [3–6]. The direct effects of local anesthesia on tumour cells are through voltage-gated sodium channels-dependent and -independent mechanisms [5,7]. In contrast, opioids are likely to promote tumour progression due to its ability to stimulate tumour angiogenesis and induce immunosuppression [8,9]. Given the increasing substantial evidence on the association between anaesthesia and tumour progression, understanding the potential mechanisms of anaesthesia and their interactions will contribute to the improved outcome of cancer patients. Although the effects of opioids and local anaesthetic on the tumour ⁎
progression have been extensively studied, the role of volatile anaesthesia in tumour remains largely unknown. Sevoflurane is the most widely used volatile anesthetic agent during surgery. The inhibitory effects of sevoflurane on proliferation and migration have been demonstrated in colon and lung cancer cells [10,11]. Chronic myeloid leukemia (CML) is a hematological stem cell malignancy. The CD34 stem/progenitor cells serve as a reservoir for disease relapse [12]. Importantly, CML CD34 cells are easier to isolate and maintain in in vitro culture system compared to other solid tumour stem/progenitor cells [13]. In this study, we used CML model to investigate the biological effects of sevoflurane on CML cell lines as well as stem/progenitor cells, and analysed the underlying molecular mechanism. 2. Methods 2.1. CML cell lines, patient samples and drugs Five human CML cell lines, KCL22, K562, KU812, LAMA84 and
Corresponding author. E-mail address: [email protected] (Z. Tan).
https://doi.org/10.1016/j.biopha.2018.08.063 Received 11 March 2018; Received in revised form 12 August 2018; Accepted 15 August 2018 0753-3322/ © 2018 Elsevier Masson SAS. All rights reserved.
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colonies plated.
KBM-7 (ATCC, US) were cultured in RPMI1640 medium supplemented with 10% fetal bovine serum, 1% penicillin/streptomycin and 2 mM Lglutamine. CD34 cells were obtained from tissue repository in Shenzhen Hospital of Southern Medical University. Briefly, CD34 cells were purified from bone marrow or peripheral blood mononuclear cells of blast phase-CML patients or from cord blood using CD34 MicroBead kit (Miltenyi Biotec, Germany). The peripheral blood or bone marrow were collected from either treatment-naïve patients or BCR-ABL tyrosine kinase inhibitors-treated patients. The patients’ clinical features were summarized in Supplementary Table 1. CD34 cells were cultured using StemPro complete medium (Life Technologies, CA, US) supplemented with 200 pg/ml of stem cell factor, 200 pg/ml of granulocyte-macrophage colony-stimulating factor, 200 pg/ml of macrophage inflammatory protein-1α, 1 ng/ml of granulocyte colony − stimulating factor, 50 pg/ml of leukemia inhibitory factor, 1 ng/ml of and interleukin 6. Sevoflurane was purchased from Abbott Inc. Dasatinib (LC laboratories, US), SB216763 and lithium chloride (LiCl, Sigma, US) were dissolved in DMSO.
2.6. Western blot A total of 1 × 106 cells (for all CML cell lines) were suspended in 2 ml culture medium and seeded in 6-well plate. After 1 day exposure to sevoflurane, cells were lysed using the same lysis buffer and the western blot was performed using the same protocol as described in our previous studies [15]. Antibodies used in western blots are against phospho-p38 MAPK (Thr180/Tyr182), P38, β-catenin, c-Myc and βactin (Cell Signaling, US). 2.7. Plasmid transfection Cell transfection was performed using Lipofectamine® Transfection Reagent (Thermo Scientific, US) according to the protocol provided by the manufacture. For rescue experiment, cells were transfected with vector plasmid (p-Vector) or plasmid containing β-catenin (p-Cat, a kind gift from Dr. Hui Li [16]). At 24 h post-transfection, cells were exposed to sevoflurane followed by proliferation rescue experiment. For TOPflash report assay, cells were transfected with a M50 Super 8x TOPFlash plasmid. After 24 h post-transfection, cells were exposed to sevoflurane followed by luciferase assay (Promega, US) according to manufacturer’s instructions.
2.2. CML cell exposure to sevoflurane and experimental protocol Cells were exposed to different percentage of sevoflurane/air mixture using the same experimental protocol described previously [14]. Briefly, cells in the culture plate were placed in the chamber with inlet and outlet connectors. The inlet port of the chamber was connected to the anaesthesia machine (Cicero-EM8060, Germany) and flushed with 0%, 2%, 4% or 8% sevoflurane mixed with 95% air/5% CO2 at 6 l/min through calibrated vaporizers (Draeger, Germany). Sevoflurane concentrations were monitored at the outlet port of the chamber using a ventilation monitor (PM8050, Germany). Cells were exposed to sevoflurane for 24 h and transferred to normal CO2 incubator prior to the following biological assays.
2.8. Real time RT-PCR Total RNA was prepared from 106 cells using RNeasy total RNA kits (Qiagen, Germany) and used to produce the first-strand cDNA using iScript cDNA Synthesis Kit (Bio-rad, CA). PCR was performed using the Platinum SYBR Green qPCR SuperMix-UDG (Invitrogen, US) on CFX96 RT PCR machine (Bio-rad, CA). The primers for the designated genes were listed in Supplementary Table 2. The relative mRNA expression levels of MYC, BCL9 and CYCLIN D1 were first normalized with βACTIN levels and then calculated relative to their treatment controls.
2.3. Proliferation assay After 1 day exposure to sevoflurane, a total of 1 × 104 cells (for all CML cell lines) from different treatment groups were suspended in 200 μl culture medium, seeded in 96-well plate and transferred to normal CO2 incubator for 2 days. Cell proliferative activity was measured by using CellTiter 96® Aqueous One Solution Cell Proliferation Assay kit (Promega, US) and BrdU Cell Proliferation Assay Kit (Cell Signaling Inc, US) according to manufacturer’s instruction.
2.9. Statistical analyses All experiments were repeated three times. The data are expressed as mean ± S.D. For statistical analysis, Prism version 6.0 (GraphPad Inc., USA) was used. For generation of related graphs, Microsoft Office related software (Microsoft Inc. USA) was used. For quantification of western blot bands, ImageJ software was used. A paired Student’s t-test was applied to determine statistical significance with p < 0.05.
2.4. Annexin V staining and flow cytometry After 1 day exposure to sevoflurane, a total of 1 × 105 cells (for all CML cell lines) were suspended in 1 ml culture medium, seeded in 12well plate and transferred to normal CO2 incubator for another 1 day. Cells were stained with ANNEXIN V-FITC / 7-AAD (Beckman Coulter, France). The percentage of Annexin V staining was measured by flow cytometry on MACSQuant® Analyzer (Miltenyl Biotec, US).
3. Results 3.1. Sevoflurane inhibits proliferation without affecting viability in CML cells We firstly investigated the biological effects of sevoflurane on CML cell growth and viability after 24 h treatment at different concentrations. We tested a panel of human CML cell lines with different cellular origin and genetic profiling that represent in vitro CML cell model [17]. We show that sevoflurane at 2%, 4% and 8% dose-dependently inhibits proliferation of all tested CML cell lines (Fig. 1A). Notably, LAMA84 is the most sensitive CML cell line to sevoflurane exposure compared with other cell lines such as KCL22, KBM-7, K562 and KU812 (Fig. 1A). To further confirm the anti-proliferative effect of sevoflurane in CML cells, we performed BrdU and cell cycle analysis after sevoflurane exposure. We found that sevoflurane dose-dependently decreased BrdU incorporation in CML cells (Fig. 1B). Cell cycle analysis indicated that sevoflurane inhibited CML cell proliferation via arresting cell cycle at G2/M (Fig. 1C). In contrast, exposure of sevoflurane at 2% and 4% does not affect CML cell viability as assessed by the flow cytometry of apoptotic marker Annexin V (Fig. 1D). Sevoflurane at 8% significantly
2.5. Colony formation and self-replating assays For colony formation assay, 1 ml of HSC-CFU complete w/o Epo methylcellulose medium (Cat No. 130-091-277, Miltenyi Biotec, Germany) mixture containing 1 × 103 CML CD34 cells (after 1 day exposure to sevoflurane) were plated onto 6-well-plate. After 10–14 days, colonies were visualized and the number of colonies was counted under light microscopy (Carl Zeiss, Germany). For serial replating assay, individual colonies formed in CFU assay were picked and replated in HSC-CFU complete w/o Epo methylcellulose medium in a 96well-plate. After 2-week incubation, wells were scored as positive or negative for the presence or absence of colonies. Three rounds of serial replating were performed. Serial replating capacity is determined by the percentage of final number of positive wells among total number of 1295
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Fig. 1. Sevoflurane significantly inhibits growth in multiple CML cell lines without affecting viability. (A) Sevoflurane significantly inhibits proliferation in CML cell lines: K562, KCL22, LAMA84, KU812 and KBM7. (B) Sevoflurane significantly inhibits BrdU incorporation in CML cell lines. (C) Sevoflurane increases G2/M phase in CML cell lines. The cell cycle was assessed by staining cells with Propidium iodide (PI) and followed by flow cytometry. (D) Sevoflurane at 2% and 4% does not affect viability in CML cell lines. *p < 0.05, compared to control.
3.2. Sevoflurane inhibits proliferation, differentiation and self-renewal capacity in CML CD34 stem/progenitor cell
but marginally increases Annexin V staining in LAMA84 and KU812 but not K562, KCL22 and KBM-7 (Fig. 1D). We further investigated whether sevoflurane affects the efficacy of standard therapeutic agent for CML treatment. We designed combination studies using dasatinib and sevoflurane at concentration that has mild inhibitory effects to CML as a single drug alone. Sevoflurane at 2% was used in the combination studies for the proliferation assay and sevoflurane at 8% was used in combination studies for the viability assay. We show that sevoflurane significantly enhances the effects of dasatinib (1 nM) in inhibiting proliferation in CML cells (Fig. 2A). In addition, combination of sevoflurane and dasatinib (1 nM) does not result in better efficacy than dasatinib alone in decreasing viability (Fig. 2B). Our results demonstrate that the predominant effect of sevoflurane on CML cells is growth inhibition.
CML is a malignance of hematopoietic stem/progenitor cells which have abilities to proliferate, differentiate and self-renew. We further examined whether sevoflurane affects CML stem/progenitor properties. We obtained CD34 cells from 10 patients with blast-phase CML and performed colony formation and serial replating assays (Supplementary Table 1). We show that sevoflurane significantly inhibits colony formation in CML CD34 cells and augments dasatinib efficacy (Fig. 3A and B), demonstrating the decreased proliferation and differentiation by sevoflurane. In addition, sevoflurane decreases the self-renewal ability of CML CD34 cells and completely abolishes the self-renewal ability during the 3rd round of replating as a single agent (Fig. 3C). We have performed colony formation and serial replating assays using cord
Fig. 2. Sevoflurane significantly enhances dasatinib’s effects in multiple CML cell lines without affecting viability. Sevoflurane significantly enhances the antiproliferative (A) but not pro-apoptotic (B) effects of dasatinib (1 nM) in CML cells. Sevoflurane at 2% was used in the combination studies for the proliferation assay and sevoflurane at 8% was used in combination studies for the viability assay. DMSO (final concentration 0.5%) was used as control. *p < 0.05, compared to single drug alone. 1296
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Fig. 3. Sevoflurane significantly inhibits colony formation and self-renewal capacity of CML CD34 stem/progenitor cells, and enhances dasatinib’s effects. Sevoflurane significantly inhibits colony formation (A) and decreases self-renewal capacity (B) of CML CD34 cells. The combination of sevoflurane (2%) and dasatinib is significantly more effective in inhibiting colony formation compared to dasatinib alone. Results shown are the percentage of the number of positive wells relative to the total number of colonies replated for the serial replating assays. (C) Sevoflurane does not affect viability of CML CD34 cells as shown by the percentage of Annexin V. Combination of sevoflurane and dasatinib does not result in less viability than dasatinib alone. Dasatinib at 50 nM was used for combination studies. Graphs presented are mean of the results obtained from ten patient-derived CML samples. *p < 0.05, compared to control or single drug alone.
Fig. 4. The effects of sevoflurane in cord blood (CB) CD34 cells. Sevoflurane at 8% but not 2% or 4% significantly inhibits colony formation (A) and self-renewal (B). The graph is obtained from three independent experiments. *, P < 0.05, compared to control.
3.3. Sevoflurane inhibits Wnt/β-catenin signaling in CML cells
blood (CB) CD34 cells. Interestingly, sevoflurane at 2% and 4% does not affect CB CD34 cell colony formation and self-renewal (Fig. 4). Sevoflurane at 8% significantly inhibits CB CD34 colony formation and selfrenewal, but to a lesser extent than in CML CD34 cells (Fig. 4). Importantly, combination of sevoflurane and dasatinib achieves better efficacy and completely abolishes the self-renewal ability during the 2nd round of replating (Fig. 3C). Consistent with the data obtained from cell lines, sevoflurane does not affect CML CD34 cell viability (Fig. 3D).
Activation of Wnt/β-catenin in CML stem/progenitors has been shown to play essential roles in CML progression via enhancing the selfrenewal activity and leukemic potential of these cells [12]. Given our observation that sevoflurane negatively targets CML stem/progenitor cell properties, we investigated the Wnt/β-catenin signaling in CML cells exposed to sevoflurane. We also examined p38 mitogen-activated protein kinases (p38 MAPK) phosphorylation level since sevoflurane has been reported to influence p38 MAPK activity in lung cancer and 1297
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Fig. 5. Sevoflurane decreases β-catenin level in CML cells. Representative western blot images (A) and quantification of western blot band density using Image-J software (B and C) showing the dose-dependently decreased levels of β-catenin and c-Myc but not p-P38 MAPK in K562 and CML CD34 stem/progenitor cells (n = 3) exposed to sevoflurane. The graph is obtained from three independent experiments. *, P < 0.05, compared to control.
GSK3β inhibitor lithium chloride (LiCl) is a Wnt activator by preventing β-catenin degradation [20], LiCl completely abolished the effect of sevoflurane in decreasing β-catenin levels in CML CD34 and K562 cells (Fig. 7A–C). The addition of LiCl reverses the inhibitory effects of sevoflurane in inhibiting proliferation in K562 cells (Fig. 7D). Importantly, LiCl significantly abolishes the effects of sevoflurane in inhibiting colony formation and self-renewal in CML CD34 cells (Fig. 7E and F). We further show that SB216763, another GSK3β inhibitor [21], also reverses the inhibitory effects of sevoflurane in inhibiting proliferation in K562 cells (Fig. 8A). In addition, SB216763 significantly abolishes the effects of sevoflurane in inhibiting colony formation and self-renewal in CML CD34 cells (Fig. 8B and C, n = 3). Similarly, sevoflurane fails to decrease β-catenin levels in β-catenin-overexpressing K562 cells (Fig. 9A). In addition, β-catenin overexpression also abolishes the anti-proliferative effect of sevoflurane in K562 cells (Fig. 9B). These data clearly demonstrate that sevoflurane acts on CML cells in a β-catenin-dependent manner.
leukemia cells [11,14,18]. We demonstrate that sevoflurane does not affect phospho-p38 MAPK level in CML CD34 and K562 cells (Fig. 5A–C). However, sevoflurane decreases levels of β-catenin and its downstream effector c-Myc in CML CD34 and K562 cells in a dose-dependent manner (Fig. 5A–C). We further show that sevoflurane significantly decreases Wnt/β-catenin activities in K562 cells as assessed by TOPflash luciferase-based reporter assay (Fig. 6A). Wnt/β-catenin signalling leads to changes in transcription of a large set of genes such as MYC, BCL9 and CYCLIN D1 [19]. Consistently, Wnt/β-catenin-mediated transcription of MYC, BCL9 and CYCLIN D1 is decreased in K562 cells exposed to sevoflurane (Fig. 6B). These results indicate that sevoflurane inhibits Wnt/β-catenin signalling in CML cells. 3.4. β-Catenin overexpression abolishes the multiple effects of sevoflurane in CML cells To confirm Wnt/β-catenin inhibition as the mechanism of sevoflurane’s action, we performed rescue experiments using both pharmacological and genetic approaches to stabilize β-catenin level in sevoflurane-treated CML cells. In line with the previous reports that
4. Discussion Since the findings on the associations between anesthesia factors
Fig. 6. Sevoflurane decreases Wnt/β-catenin activities in CML cells. (A) Sevoflurane significantly inhibits TOPflash activation. K562 cells transfected with TOPflash plasmid were exposed to sevoflurane as indicated. Graph represents mean ± S.D of TOPflash signal relative to control. RLU, relative light units. (B) Sevoflurane significantly reduces levels of MYC, BCL9 and CYCLIN D1 in K562 cells. *p < 0.05, compared to control. 1298
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Fig. 7. Wnt/β-catenin activator LiCl significantly reverses the effects of sevoflurane in CML cells. Representative western blot image (A) and quantification using Image J software (B and C) showing that the addition of LiCl (50 mM) reverses the decreased β-catenin level in CML CD34 and K562 cells. LiCl significantly reverses the decreased proliferation (D), colony formation (E) and self-renewal capacity (F) by sevoflurane in CML CD34 (n = 5) or K562 cells. *p < 0.05, compared to control.
Fig. 8. Wnt/β-catenin activator SB216763 significantly reverses the effects of sevoflurane in CML cells. Addition of SB216763 (10 mM) significantly reverses the decreased proliferation (A), colony formation (B) and self-renewal capacity (C) by sevoflurane in K562 or CML CD34 (n = 3) cells. *, P < 0.05, compared to control. 1299
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Fig. 9. Overexpression of β-catenin significantly reverses the effects of sevoflurane in CML cells. Overexpression of β-catenin reverses the decreased β-catenin level (A) and proliferation (B) by sevoflurane in K562 cells. *p < 0.05, compared to control.
surgery and improve overall survival in cancer patients. A significant finding of our work is the identification of Wnt/β-catenin inhibition as the molecular mechanism of sevoflurane in CML cells. Many studies demonstrate that sevoflurane acts on tumour cells via modulating p38 MAPK activity [11,14,18]. We show that sevoflurane targets CML via inhibiting Wnt/β-catenin in a p38 MAPK-independent manner (Figs. 5–9). Our findings on the molecular mechanisms of sevoflurane’s action are supported by the previous work that sevoflurane suppresses Wnt/β-catenin in mouse neural progenitor [29,30]. It is known that Wnt/β-catenin is essential to maintain the stemness and expansion of not only CML CD34 cells but also stem cells of other cellular origin [31]. The ability of sevoflurane in inhibiting Wnt/β-catenin suggests that sevoflurane might have direct effects on other types of tumour and normal stem cells. In conclusion, our work using cell lines and primary patient samples demonstrate the inhibitory effects of sevoflurane on the various biological aspects of CML cells. Our work also demonstrates the synergy between sevoflurane and dasatinib in CML and adds Wnt/ β-catenin to the list of sevoflurane’s molecular targets.
and tumour progression, understating the direct effects of anesthetics on various type of tumours has gained increasingly attention. Most studies focus on local anesthetics (eg, ropivacaine and lidocaine), intravenous anesthetics (eg, propofol) and opioids (eg, morphine), and demonstrate their possible roles on the multiple aspects of tumour biology, such as growth, survival, invasion, angiogenesis and chemoresistance [7,9,22–24]. In lines with these efforts, our previous studies also show that propofol enhances imatinib’s inhibitory effects in CML through Akt/mTOR suppression [15]. Notably, very few available information exists regarding the direct effects of volatile anesthetics in tumour. In this work, we systematically investigated the effects of sevoflurane, the most commonly used volatile anesthetics, on CML cell line and stem/progenitor cells. Our findings indicate the inhibitory effects of sevoflurane on the growth, differentiation and self-renewal of leukemia stem/progenitor cells. We demonstrate the dose-dependent inhibition of growth in a panel of cell lines presenting differentiated CML cells whereas the survival is largely unaffected by sevoflurane (Fig. 1), suggesting the predominant inhibitory effects of sevoflurane in growth regardless of leukemia cell origin and genetic profiling. We further demonstrate the inhibitory effects of sevoflurane on CML CD34 stem/progenitor cell growth, differentiation and self-renewal but not survival (Fig. 3). Our findings on the inability of sevoflurane in inducing leukemia cell death together with Martin et al’s work that sevoflurane up to 8% marginally induces apoptosis in Jurkat cells [18] indicate that sevoflurane is unlikely to affect tumour cell survival. In contrast, several studies have demonstrated and confirmed the inhibitory effects of sevoflurane on growth, migration and invasion in different types of tumours, such as lung cancer and glioblastoma cells [11,25,26], which is further supported by our findings that sevoflurane inhibits growth of leukemia stem/progenitor cells. Apart from inhibition of growth and migration, our work is the first to demonstrate that sevoflurane inhibits leukemia stem/progenitor cell differentiation and self-renewal (Fig. 3B and C). Interestingly, Shi et al’s work show that sevoflurane promotes the expansion of glioma stem cells through activation of hypoxia-inducible factors in vitro [27]. The differences in the sevoflurane concentrations, treatment duration and cell models might explain the different effects of sevoflurane on stem cell proliferation. In addition, our findings and Shi et al’s work also suggest that some effects of sevoflurane on tumour cells might be tumour type-specific. To simulate the clinical settings, we analysed the combinatory effects of sevoflurane with standard therapeutic agent in CML. We show that combination of sevoflurane with dasatinib results in significant greater efficacy than dasatinib alone in inhibiting growth, differentiation and self-renewal capacity of CML cell line and stem/progenitor cells (Figs. 2 and 3). This is supported by the previous findings on the growth-inhibitory and invasion-inhibitory synergy between sevoflurane and cisplatin in lung adenocarcinoma A549 cells [28]. The potent inhibitory effects of sevoflurane alone and its synergy with chemotherapeutic agents on tumour differentiated as well as stem/initiating cells suggest that sevoflurane may suppress the tumour recurrence after
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Volatile anesthetics sevoflurane targets leukemia stem/progenitor cells via Wnt/β-catenin inhibition
T
Xuguang Ruana, Weihang Jianga, Pingrui Chenga, Lingyan Huanga, Xuelan Lia, Yingyi Hea, ⁎ Minyi Maia, Zhimin Tanb, a b
Department of Anesthesiology, Panyu Central Hospital, Guangzhou, China Department of Anesthesiology, Shenzhen Hospital, Southern Medical University, Xinhu Road 1333, Bao’an District, Shenzhen, China
A R T I C LE I N FO
A B S T R A C T
Keywords: Sevoflurane Volatile anesthetics Wnt/β-catenin Stem/progenitors Leukemia
Most of the studies regarding the direct effect of anesthetics on tumour cells are focused on opioids and voltagegated sodium channels. Little is known on the effect of volatile anesthetics on tumour progression. In this study, we show that sevoflurane, a volatile anesthetic, negatively affects chronic myeloid leukemia (CML) CD34 stem/ progenitor cells’ biological properties. Sevoflurane significantly inhibits the growth of a panel of CML cell lines in a dose-dependent manner without affecting their survival. It also inhibits proliferation, differentiation and selfrenewal capacities but not survival of CML CD34 cells. In addition, sevoflurane significantly augments dasatinib’s efficacy in CML cell lines and stem/progenitors. Mechanistically, sevoflurane dose-dependently decreases levels of β-catenin and c-Myc but not phospho-P38 MAPK in K562 and CML CD34 cells. The decreased Wnt/ βcatenin activity and the reduced levels of Wnt/β-catenin-targeted transcriptions are observed in CML cells exposed to sevoflurane. The complete rescue of the inhibitory effects of sevoflurane in K562 and CML CD34 cells by β-catenin stabilization using both genetic and pharmacological approaches further demonstrates that sevoflurane acts on CML cells via a β-catenin-dependent manner. Our results clearly show the direct and negative effects of sevoflurane on the leukemia cell lines as well as leukemia stem/progenitors. Our findings also reveal Wnt/β-catenin as the target of volatile anesthetics.
1. Introduction A number of retrospective studies suggest that the type of anesthetics as well as anesthetic techniques chosen for cancer patients during surgery or chronic pain control management have potential influence on the long-term outcome of the disease [1]. For example, local anaesthesia is associated with reduced cancer recurrence and improved overall survival [2]. Studies using pre-clinical models demonstrate that local anaesthesia including ropivacaine, lidocaine and bupivacaine, inhibit various tumour growth and metastasis, and induce tumour cell death [3–6]. The direct effects of local anesthesia on tumour cells are through voltage-gated sodium channels-dependent and -independent mechanisms [5,7]. In contrast, opioids are likely to promote tumour progression due to its ability to stimulate tumour angiogenesis and induce immunosuppression [8,9]. Given the increasing substantial evidence on the association between anaesthesia and tumour progression, understanding the potential mechanisms of anaesthesia and their interactions will contribute to the improved outcome of cancer patients. Although the effects of opioids and local anaesthetic on the tumour ⁎
progression have been extensively studied, the role of volatile anaesthesia in tumour remains largely unknown. Sevoflurane is the most widely used volatile anesthetic agent during surgery. The inhibitory effects of sevoflurane on proliferation and migration have been demonstrated in colon and lung cancer cells [10,11]. Chronic myeloid leukemia (CML) is a hematological stem cell malignancy. The CD34 stem/progenitor cells serve as a reservoir for disease relapse [12]. Importantly, CML CD34 cells are easier to isolate and maintain in in vitro culture system compared to other solid tumour stem/progenitor cells [13]. In this study, we used CML model to investigate the biological effects of sevoflurane on CML cell lines as well as stem/progenitor cells, and analysed the underlying molecular mechanism. 2. Methods 2.1. CML cell lines, patient samples and drugs Five human CML cell lines, KCL22, K562, KU812, LAMA84 and
Corresponding author. E-mail address: [email protected] (Z. Tan).
https://doi.org/10.1016/j.biopha.2018.08.063 Received 11 March 2018; Received in revised form 12 August 2018; Accepted 15 August 2018 0753-3322/ © 2018 Elsevier Masson SAS. All rights reserved.
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colonies plated.
KBM-7 (ATCC, US) were cultured in RPMI1640 medium supplemented with 10% fetal bovine serum, 1% penicillin/streptomycin and 2 mM Lglutamine. CD34 cells were obtained from tissue repository in Shenzhen Hospital of Southern Medical University. Briefly, CD34 cells were purified from bone marrow or peripheral blood mononuclear cells of blast phase-CML patients or from cord blood using CD34 MicroBead kit (Miltenyi Biotec, Germany). The peripheral blood or bone marrow were collected from either treatment-naïve patients or BCR-ABL tyrosine kinase inhibitors-treated patients. The patients’ clinical features were summarized in Supplementary Table 1. CD34 cells were cultured using StemPro complete medium (Life Technologies, CA, US) supplemented with 200 pg/ml of stem cell factor, 200 pg/ml of granulocyte-macrophage colony-stimulating factor, 200 pg/ml of macrophage inflammatory protein-1α, 1 ng/ml of granulocyte colony − stimulating factor, 50 pg/ml of leukemia inhibitory factor, 1 ng/ml of and interleukin 6. Sevoflurane was purchased from Abbott Inc. Dasatinib (LC laboratories, US), SB216763 and lithium chloride (LiCl, Sigma, US) were dissolved in DMSO.
2.6. Western blot A total of 1 × 106 cells (for all CML cell lines) were suspended in 2 ml culture medium and seeded in 6-well plate. After 1 day exposure to sevoflurane, cells were lysed using the same lysis buffer and the western blot was performed using the same protocol as described in our previous studies [15]. Antibodies used in western blots are against phospho-p38 MAPK (Thr180/Tyr182), P38, β-catenin, c-Myc and βactin (Cell Signaling, US). 2.7. Plasmid transfection Cell transfection was performed using Lipofectamine® Transfection Reagent (Thermo Scientific, US) according to the protocol provided by the manufacture. For rescue experiment, cells were transfected with vector plasmid (p-Vector) or plasmid containing β-catenin (p-Cat, a kind gift from Dr. Hui Li [16]). At 24 h post-transfection, cells were exposed to sevoflurane followed by proliferation rescue experiment. For TOPflash report assay, cells were transfected with a M50 Super 8x TOPFlash plasmid. After 24 h post-transfection, cells were exposed to sevoflurane followed by luciferase assay (Promega, US) according to manufacturer’s instructions.
2.2. CML cell exposure to sevoflurane and experimental protocol Cells were exposed to different percentage of sevoflurane/air mixture using the same experimental protocol described previously [14]. Briefly, cells in the culture plate were placed in the chamber with inlet and outlet connectors. The inlet port of the chamber was connected to the anaesthesia machine (Cicero-EM8060, Germany) and flushed with 0%, 2%, 4% or 8% sevoflurane mixed with 95% air/5% CO2 at 6 l/min through calibrated vaporizers (Draeger, Germany). Sevoflurane concentrations were monitored at the outlet port of the chamber using a ventilation monitor (PM8050, Germany). Cells were exposed to sevoflurane for 24 h and transferred to normal CO2 incubator prior to the following biological assays.
2.8. Real time RT-PCR Total RNA was prepared from 106 cells using RNeasy total RNA kits (Qiagen, Germany) and used to produce the first-strand cDNA using iScript cDNA Synthesis Kit (Bio-rad, CA). PCR was performed using the Platinum SYBR Green qPCR SuperMix-UDG (Invitrogen, US) on CFX96 RT PCR machine (Bio-rad, CA). The primers for the designated genes were listed in Supplementary Table 2. The relative mRNA expression levels of MYC, BCL9 and CYCLIN D1 were first normalized with βACTIN levels and then calculated relative to their treatment controls.
2.3. Proliferation assay After 1 day exposure to sevoflurane, a total of 1 × 104 cells (for all CML cell lines) from different treatment groups were suspended in 200 μl culture medium, seeded in 96-well plate and transferred to normal CO2 incubator for 2 days. Cell proliferative activity was measured by using CellTiter 96® Aqueous One Solution Cell Proliferation Assay kit (Promega, US) and BrdU Cell Proliferation Assay Kit (Cell Signaling Inc, US) according to manufacturer’s instruction.
2.9. Statistical analyses All experiments were repeated three times. The data are expressed as mean ± S.D. For statistical analysis, Prism version 6.0 (GraphPad Inc., USA) was used. For generation of related graphs, Microsoft Office related software (Microsoft Inc. USA) was used. For quantification of western blot bands, ImageJ software was used. A paired Student’s t-test was applied to determine statistical significance with p < 0.05.
2.4. Annexin V staining and flow cytometry After 1 day exposure to sevoflurane, a total of 1 × 105 cells (for all CML cell lines) were suspended in 1 ml culture medium, seeded in 12well plate and transferred to normal CO2 incubator for another 1 day. Cells were stained with ANNEXIN V-FITC / 7-AAD (Beckman Coulter, France). The percentage of Annexin V staining was measured by flow cytometry on MACSQuant® Analyzer (Miltenyl Biotec, US).
3. Results 3.1. Sevoflurane inhibits proliferation without affecting viability in CML cells We firstly investigated the biological effects of sevoflurane on CML cell growth and viability after 24 h treatment at different concentrations. We tested a panel of human CML cell lines with different cellular origin and genetic profiling that represent in vitro CML cell model [17]. We show that sevoflurane at 2%, 4% and 8% dose-dependently inhibits proliferation of all tested CML cell lines (Fig. 1A). Notably, LAMA84 is the most sensitive CML cell line to sevoflurane exposure compared with other cell lines such as KCL22, KBM-7, K562 and KU812 (Fig. 1A). To further confirm the anti-proliferative effect of sevoflurane in CML cells, we performed BrdU and cell cycle analysis after sevoflurane exposure. We found that sevoflurane dose-dependently decreased BrdU incorporation in CML cells (Fig. 1B). Cell cycle analysis indicated that sevoflurane inhibited CML cell proliferation via arresting cell cycle at G2/M (Fig. 1C). In contrast, exposure of sevoflurane at 2% and 4% does not affect CML cell viability as assessed by the flow cytometry of apoptotic marker Annexin V (Fig. 1D). Sevoflurane at 8% significantly
2.5. Colony formation and self-replating assays For colony formation assay, 1 ml of HSC-CFU complete w/o Epo methylcellulose medium (Cat No. 130-091-277, Miltenyi Biotec, Germany) mixture containing 1 × 103 CML CD34 cells (after 1 day exposure to sevoflurane) were plated onto 6-well-plate. After 10–14 days, colonies were visualized and the number of colonies was counted under light microscopy (Carl Zeiss, Germany). For serial replating assay, individual colonies formed in CFU assay were picked and replated in HSC-CFU complete w/o Epo methylcellulose medium in a 96well-plate. After 2-week incubation, wells were scored as positive or negative for the presence or absence of colonies. Three rounds of serial replating were performed. Serial replating capacity is determined by the percentage of final number of positive wells among total number of 1295
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Fig. 1. Sevoflurane significantly inhibits growth in multiple CML cell lines without affecting viability. (A) Sevoflurane significantly inhibits proliferation in CML cell lines: K562, KCL22, LAMA84, KU812 and KBM7. (B) Sevoflurane significantly inhibits BrdU incorporation in CML cell lines. (C) Sevoflurane increases G2/M phase in CML cell lines. The cell cycle was assessed by staining cells with Propidium iodide (PI) and followed by flow cytometry. (D) Sevoflurane at 2% and 4% does not affect viability in CML cell lines. *p < 0.05, compared to control.
3.2. Sevoflurane inhibits proliferation, differentiation and self-renewal capacity in CML CD34 stem/progenitor cell
but marginally increases Annexin V staining in LAMA84 and KU812 but not K562, KCL22 and KBM-7 (Fig. 1D). We further investigated whether sevoflurane affects the efficacy of standard therapeutic agent for CML treatment. We designed combination studies using dasatinib and sevoflurane at concentration that has mild inhibitory effects to CML as a single drug alone. Sevoflurane at 2% was used in the combination studies for the proliferation assay and sevoflurane at 8% was used in combination studies for the viability assay. We show that sevoflurane significantly enhances the effects of dasatinib (1 nM) in inhibiting proliferation in CML cells (Fig. 2A). In addition, combination of sevoflurane and dasatinib (1 nM) does not result in better efficacy than dasatinib alone in decreasing viability (Fig. 2B). Our results demonstrate that the predominant effect of sevoflurane on CML cells is growth inhibition.
CML is a malignance of hematopoietic stem/progenitor cells which have abilities to proliferate, differentiate and self-renew. We further examined whether sevoflurane affects CML stem/progenitor properties. We obtained CD34 cells from 10 patients with blast-phase CML and performed colony formation and serial replating assays (Supplementary Table 1). We show that sevoflurane significantly inhibits colony formation in CML CD34 cells and augments dasatinib efficacy (Fig. 3A and B), demonstrating the decreased proliferation and differentiation by sevoflurane. In addition, sevoflurane decreases the self-renewal ability of CML CD34 cells and completely abolishes the self-renewal ability during the 3rd round of replating as a single agent (Fig. 3C). We have performed colony formation and serial replating assays using cord
Fig. 2. Sevoflurane significantly enhances dasatinib’s effects in multiple CML cell lines without affecting viability. Sevoflurane significantly enhances the antiproliferative (A) but not pro-apoptotic (B) effects of dasatinib (1 nM) in CML cells. Sevoflurane at 2% was used in the combination studies for the proliferation assay and sevoflurane at 8% was used in combination studies for the viability assay. DMSO (final concentration 0.5%) was used as control. *p < 0.05, compared to single drug alone. 1296
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Fig. 3. Sevoflurane significantly inhibits colony formation and self-renewal capacity of CML CD34 stem/progenitor cells, and enhances dasatinib’s effects. Sevoflurane significantly inhibits colony formation (A) and decreases self-renewal capacity (B) of CML CD34 cells. The combination of sevoflurane (2%) and dasatinib is significantly more effective in inhibiting colony formation compared to dasatinib alone. Results shown are the percentage of the number of positive wells relative to the total number of colonies replated for the serial replating assays. (C) Sevoflurane does not affect viability of CML CD34 cells as shown by the percentage of Annexin V. Combination of sevoflurane and dasatinib does not result in less viability than dasatinib alone. Dasatinib at 50 nM was used for combination studies. Graphs presented are mean of the results obtained from ten patient-derived CML samples. *p < 0.05, compared to control or single drug alone.
Fig. 4. The effects of sevoflurane in cord blood (CB) CD34 cells. Sevoflurane at 8% but not 2% or 4% significantly inhibits colony formation (A) and self-renewal (B). The graph is obtained from three independent experiments. *, P < 0.05, compared to control.
3.3. Sevoflurane inhibits Wnt/β-catenin signaling in CML cells
blood (CB) CD34 cells. Interestingly, sevoflurane at 2% and 4% does not affect CB CD34 cell colony formation and self-renewal (Fig. 4). Sevoflurane at 8% significantly inhibits CB CD34 colony formation and selfrenewal, but to a lesser extent than in CML CD34 cells (Fig. 4). Importantly, combination of sevoflurane and dasatinib achieves better efficacy and completely abolishes the self-renewal ability during the 2nd round of replating (Fig. 3C). Consistent with the data obtained from cell lines, sevoflurane does not affect CML CD34 cell viability (Fig. 3D).
Activation of Wnt/β-catenin in CML stem/progenitors has been shown to play essential roles in CML progression via enhancing the selfrenewal activity and leukemic potential of these cells [12]. Given our observation that sevoflurane negatively targets CML stem/progenitor cell properties, we investigated the Wnt/β-catenin signaling in CML cells exposed to sevoflurane. We also examined p38 mitogen-activated protein kinases (p38 MAPK) phosphorylation level since sevoflurane has been reported to influence p38 MAPK activity in lung cancer and 1297
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Fig. 5. Sevoflurane decreases β-catenin level in CML cells. Representative western blot images (A) and quantification of western blot band density using Image-J software (B and C) showing the dose-dependently decreased levels of β-catenin and c-Myc but not p-P38 MAPK in K562 and CML CD34 stem/progenitor cells (n = 3) exposed to sevoflurane. The graph is obtained from three independent experiments. *, P < 0.05, compared to control.
GSK3β inhibitor lithium chloride (LiCl) is a Wnt activator by preventing β-catenin degradation [20], LiCl completely abolished the effect of sevoflurane in decreasing β-catenin levels in CML CD34 and K562 cells (Fig. 7A–C). The addition of LiCl reverses the inhibitory effects of sevoflurane in inhibiting proliferation in K562 cells (Fig. 7D). Importantly, LiCl significantly abolishes the effects of sevoflurane in inhibiting colony formation and self-renewal in CML CD34 cells (Fig. 7E and F). We further show that SB216763, another GSK3β inhibitor [21], also reverses the inhibitory effects of sevoflurane in inhibiting proliferation in K562 cells (Fig. 8A). In addition, SB216763 significantly abolishes the effects of sevoflurane in inhibiting colony formation and self-renewal in CML CD34 cells (Fig. 8B and C, n = 3). Similarly, sevoflurane fails to decrease β-catenin levels in β-catenin-overexpressing K562 cells (Fig. 9A). In addition, β-catenin overexpression also abolishes the anti-proliferative effect of sevoflurane in K562 cells (Fig. 9B). These data clearly demonstrate that sevoflurane acts on CML cells in a β-catenin-dependent manner.
leukemia cells [11,14,18]. We demonstrate that sevoflurane does not affect phospho-p38 MAPK level in CML CD34 and K562 cells (Fig. 5A–C). However, sevoflurane decreases levels of β-catenin and its downstream effector c-Myc in CML CD34 and K562 cells in a dose-dependent manner (Fig. 5A–C). We further show that sevoflurane significantly decreases Wnt/β-catenin activities in K562 cells as assessed by TOPflash luciferase-based reporter assay (Fig. 6A). Wnt/β-catenin signalling leads to changes in transcription of a large set of genes such as MYC, BCL9 and CYCLIN D1 [19]. Consistently, Wnt/β-catenin-mediated transcription of MYC, BCL9 and CYCLIN D1 is decreased in K562 cells exposed to sevoflurane (Fig. 6B). These results indicate that sevoflurane inhibits Wnt/β-catenin signalling in CML cells. 3.4. β-Catenin overexpression abolishes the multiple effects of sevoflurane in CML cells To confirm Wnt/β-catenin inhibition as the mechanism of sevoflurane’s action, we performed rescue experiments using both pharmacological and genetic approaches to stabilize β-catenin level in sevoflurane-treated CML cells. In line with the previous reports that
4. Discussion Since the findings on the associations between anesthesia factors
Fig. 6. Sevoflurane decreases Wnt/β-catenin activities in CML cells. (A) Sevoflurane significantly inhibits TOPflash activation. K562 cells transfected with TOPflash plasmid were exposed to sevoflurane as indicated. Graph represents mean ± S.D of TOPflash signal relative to control. RLU, relative light units. (B) Sevoflurane significantly reduces levels of MYC, BCL9 and CYCLIN D1 in K562 cells. *p < 0.05, compared to control. 1298
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Fig. 7. Wnt/β-catenin activator LiCl significantly reverses the effects of sevoflurane in CML cells. Representative western blot image (A) and quantification using Image J software (B and C) showing that the addition of LiCl (50 mM) reverses the decreased β-catenin level in CML CD34 and K562 cells. LiCl significantly reverses the decreased proliferation (D), colony formation (E) and self-renewal capacity (F) by sevoflurane in CML CD34 (n = 5) or K562 cells. *p < 0.05, compared to control.
Fig. 8. Wnt/β-catenin activator SB216763 significantly reverses the effects of sevoflurane in CML cells. Addition of SB216763 (10 mM) significantly reverses the decreased proliferation (A), colony formation (B) and self-renewal capacity (C) by sevoflurane in K562 or CML CD34 (n = 3) cells. *, P < 0.05, compared to control. 1299
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Fig. 9. Overexpression of β-catenin significantly reverses the effects of sevoflurane in CML cells. Overexpression of β-catenin reverses the decreased β-catenin level (A) and proliferation (B) by sevoflurane in K562 cells. *p < 0.05, compared to control.
surgery and improve overall survival in cancer patients. A significant finding of our work is the identification of Wnt/β-catenin inhibition as the molecular mechanism of sevoflurane in CML cells. Many studies demonstrate that sevoflurane acts on tumour cells via modulating p38 MAPK activity [11,14,18]. We show that sevoflurane targets CML via inhibiting Wnt/β-catenin in a p38 MAPK-independent manner (Figs. 5–9). Our findings on the molecular mechanisms of sevoflurane’s action are supported by the previous work that sevoflurane suppresses Wnt/β-catenin in mouse neural progenitor [29,30]. It is known that Wnt/β-catenin is essential to maintain the stemness and expansion of not only CML CD34 cells but also stem cells of other cellular origin [31]. The ability of sevoflurane in inhibiting Wnt/β-catenin suggests that sevoflurane might have direct effects on other types of tumour and normal stem cells. In conclusion, our work using cell lines and primary patient samples demonstrate the inhibitory effects of sevoflurane on the various biological aspects of CML cells. Our work also demonstrates the synergy between sevoflurane and dasatinib in CML and adds Wnt/ β-catenin to the list of sevoflurane’s molecular targets.
and tumour progression, understating the direct effects of anesthetics on various type of tumours has gained increasingly attention. Most studies focus on local anesthetics (eg, ropivacaine and lidocaine), intravenous anesthetics (eg, propofol) and opioids (eg, morphine), and demonstrate their possible roles on the multiple aspects of tumour biology, such as growth, survival, invasion, angiogenesis and chemoresistance [7,9,22–24]. In lines with these efforts, our previous studies also show that propofol enhances imatinib’s inhibitory effects in CML through Akt/mTOR suppression [15]. Notably, very few available information exists regarding the direct effects of volatile anesthetics in tumour. In this work, we systematically investigated the effects of sevoflurane, the most commonly used volatile anesthetics, on CML cell line and stem/progenitor cells. Our findings indicate the inhibitory effects of sevoflurane on the growth, differentiation and self-renewal of leukemia stem/progenitor cells. We demonstrate the dose-dependent inhibition of growth in a panel of cell lines presenting differentiated CML cells whereas the survival is largely unaffected by sevoflurane (Fig. 1), suggesting the predominant inhibitory effects of sevoflurane in growth regardless of leukemia cell origin and genetic profiling. We further demonstrate the inhibitory effects of sevoflurane on CML CD34 stem/progenitor cell growth, differentiation and self-renewal but not survival (Fig. 3). Our findings on the inability of sevoflurane in inducing leukemia cell death together with Martin et al’s work that sevoflurane up to 8% marginally induces apoptosis in Jurkat cells [18] indicate that sevoflurane is unlikely to affect tumour cell survival. In contrast, several studies have demonstrated and confirmed the inhibitory effects of sevoflurane on growth, migration and invasion in different types of tumours, such as lung cancer and glioblastoma cells [11,25,26], which is further supported by our findings that sevoflurane inhibits growth of leukemia stem/progenitor cells. Apart from inhibition of growth and migration, our work is the first to demonstrate that sevoflurane inhibits leukemia stem/progenitor cell differentiation and self-renewal (Fig. 3B and C). Interestingly, Shi et al’s work show that sevoflurane promotes the expansion of glioma stem cells through activation of hypoxia-inducible factors in vitro [27]. The differences in the sevoflurane concentrations, treatment duration and cell models might explain the different effects of sevoflurane on stem cell proliferation. In addition, our findings and Shi et al’s work also suggest that some effects of sevoflurane on tumour cells might be tumour type-specific. To simulate the clinical settings, we analysed the combinatory effects of sevoflurane with standard therapeutic agent in CML. We show that combination of sevoflurane with dasatinib results in significant greater efficacy than dasatinib alone in inhibiting growth, differentiation and self-renewal capacity of CML cell line and stem/progenitor cells (Figs. 2 and 3). This is supported by the previous findings on the growth-inhibitory and invasion-inhibitory synergy between sevoflurane and cisplatin in lung adenocarcinoma A549 cells [28]. The potent inhibitory effects of sevoflurane alone and its synergy with chemotherapeutic agents on tumour differentiated as well as stem/initiating cells suggest that sevoflurane may suppress the tumour recurrence after
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