Background
Chronic myeloid leukemia (CML) is a hematological stem cell malignancy. The majority of CML are due to transformation of oncogene BCR-ABL and 1–2% CML are BCR-ABL negative [
1,
2]. Treatment with tyrosine kinase inhibitors (TKIs) specifically targeting BCR-ABL by binding to the ATP-binding site of Abl, such as imatinib and dasatinib, results in significant improvement in clinical responses of CML patients [
3,
4]. However, patients achieving remission with BCR-ABL TKIs continue to have molecular evidence of persistent disease and major mechanisms are due to Bcr-Abl protein overexpression and mutations [
5]. Other BCR-ABL-independent resistance mechanisms have been identified to be compensatory activation of phosphoinositide 3-kinase (PI3K)/Akt/mammalian target of rapamycin (mTOR) and Wnt/β-catenin, and suppression of protein phosphatase 2A [
6‐
8]. Therefore, identification of compounds that target the molecules involved in the resistance may provide an alternative therapeutic strategy for CML treatment.
Propofol is a general sedative reagent and commonly used for induction and maintenance of general anesthesia [
9]. It has advantages over other anesthetic drugs by protecting neuron and endothelial cells from oxidative stress and hypoxia injury [
10,
11]. Interestingly, increasing studies have demonstrated that propofol inhibits the growth, migration and invasion and induces apoptosis of tumor cells of diverse tissue origins, such as ovarian, cervix, lung and gastric-intestinal tract [
12‐
16]. The synergistic effects of propofol with conventional chemotherapeutic drugs have been demonstrated in cervical and ovarian cancer cells [
13,
17]. The mechanism of action of propofol in cancer is not completely understood and seems to be different in various tumor types. For example, it kills lung cancer cells via inducing endoplasmic reticulum stress [
16] whereas promotes cervical cancer cell apoptosis via inhibiting mTOR pathway [
18].
In this study, we examined the effect of propofol alone and its combinatory effect with BCR-ABL TKIs in CML cell lines, primary CD34 progenitor cells and xenograft mouse model. We show that propofol is effective in targeting multiple aspects of CML cells and acts synergistically with BCR-ABL TKIs in vitro and in vivo. We further show that propofol augments TKIs’ effect via suppressing Akt/mTOR signaling pathway in CML cells.
Methods
CML patient CD34 cells, cell lines and drugs
CD34 cells were obtained from tissue repository in Shenzhen Hospital of Southern Medical University and The Fifth Affiliated Hospital of Southern Medical University. Human normal bone marrow (NBM) CD34 progenitor cells were purchased from LONZA Group. CD34 cells were cultured in a serum-free medium supplemented with multiple recombinant cytokines for myelopoiesis of hematopoietic progenitor cells as previously described [
19]. Human CML cell lines (eg. K562, KU812 and KBM-7) were purchased from American Type Culture Collection and cultured in RPMI1640 medium supplemented with 10% fetal bovine serum and 2 mM L-glutamine. Dasatinib (LC laboratories, US) and propofol (Sigma, US) were reconstituted in dimethyl sulfoxide (DMSO) and imatinib (Sigma, US) was reconstituted in water.
MTS proliferation assay
Equal number of CML cells (10,000) were seeded into 96-well-plate and incubated with propofol or imatinib alone or combination of propofol and imatinib for 72 h. Cell proliferative activity was then measured by using CellTiter 96® Aqueous One Solution Cell Proliferation Assay kit (Promega, US) according to manufacture’s instruction.
Apoptosis analysis and caspase-3activity assay
CML cells (500, 000) were seeded into 12-well-plate and incubated with propofol or imatinib alone or combination of propofol and imatinib for 72 h. Apoptotic cells were labeled by Annexin V-FITC and 7-AAD staining using ANNEXIN V-FITC / 7-AAD Kit (Beckman Coulter, France). Quantification of apoptotic cells were achieved by performing flow cytometry on MACSQuant® Analyzer (Miltenyl Biotec, US). Cells were incubated with propofol for 48 h prior to caspase 3 activity assay using Caspase 3 Assay Kit (Abcam, US).
CD34 cells, HSC-CFU complete w/o Epo methylcellulose medium (Miltenyi Biotec, Germany) together with drug were mixed well and plated onto 6-well-plate. After 10–14 days, colonies were visualized under microscopy and the number of colonies was scored. Clusters with more than 100 cells were counted as a colony.
Western blot
K562 cells (2,000,000) were seeded into 6-well-plate and incubated with propofol or imatinib alone or combination of propofol and imatinib for 24 h. Treated cells were then lysed in M2 lysis buffer (20 mM Tris at pH 7, 0.5% NP-40, 250 mM NaCl, 3 mM EGTA, 3 mM EDTA, 2 mM dithiothreitol, 20 mM glycerol phosphate, proteinases inhibitor cocktail). Protein concentration was determined using bicinchoninic acid protein assay kit (Thermo Scientific, US). Equal amount of protein were resolved by SDS-PAGE and transferred onto PVDF membrane (Bio-Rad, US). The membrane was then analysed by western blot with designated primary and secondary antibodies. Signals were developed with the chemiluminescence kits (Amersham Biosciences, UK) and visualized with the Kodak Image Station.
Plasmid transfection
K562 cells were transfected by treating the cells with Lipofectamine® Transfection Reagent (Thermo Scientific, US) and 2 μg Vector, or myr Akt (constitutively active Akt) plasmid (a kind gift from Dr. Richard Roth) [
20] using the protocol provided by the manufacture. At 24 h post-transfection, cells were used for rescue experiments.
CML xenograft in SCID mouse
Animal experiments were carried out in compliance with the Fifth Affiliated Hospital of Southern Medical University. 6-week-old male NOD/SCID mice were purchased from Hunan SJA Animal Laborator Co. Ltd. K562 cells (10, 000,000) were harvested and suspended in 100 μl cold PBS. Cells were subcutaneously injected into mice flank. After development of palpable tumor, mice were randomized into four groups and treated with intraperitoneal control (80/20%, saline/DMSO), oral dasatinib, intraperitoneal propofol or combination of dasatinib and propofol daily. Tumor size were measured every 3 days. After 3 weeks treatment, mice were euthanized and tumors were weighed.
Statistical analyses
All experiments were repeated at least three times with similar results. The data are expressed as mean ± S.D. An unpaired Student’s t test was applied to determine statistical significance with p < 0.05.
Discussion
Although the inhibitory effect for an intravenous anesthetic drug propofol as an anticancer agent has been demonstrated in a panel of solid tumors [
13‐
15,
23,
29,
30], its therapeutic potential in hematological malignancies are not well understood. CML is characterized as a hematopoietic stem cell malignancy [
1]. Although imatinib or dasatinib have significantly improved clinical outcome in CML, primary resistance and leukemia relapse after an initially successful response is a major problem [
31]. This suggests that addition of drug which can synergize with TKI may be better option in CML. In this work, we addressed a most relevant issue in CML patients by evaluating propofol as a potential agent for overcoming BCR-ABL TKIs resistance. Propofol is an attractive candidate as it is available in clinical practice for the induction and maintenance of general anesthesia, and have protective effects in multiple organs and tissues from hypoxia and ischemia-reperfusion injuries [
11,
32]. In this study, we show that propofol is active against CML cells and significantly augments TKIs’ inhibitory effect via suppression Akt/mTOR signaling.
We used three cell lines: KBM7, K562 and KU812, which derived from different CML patients, to ensure the effect of propofol in CML. Propofol significantly inhibits proliferation, increases apoptosis and caspase 3 activation in all tested CML cell lines (Fig.
1a to
c). This result is consistent with the previous reports on the inhibitory effects of propofol on the tumor cell growth and survival [
13‐
15,
23,
29,
30]. In particular, propofol has been shown to activate caspase 3, 8 and 9 in acute myeloid leukemia cell HL60 [
24]. In addition, propofol significantly sensitizes CML cell in response to imatinib (Fig.
1d and e). The plasma propofol concentrations for general anesthesia are considered to be 3–6 μg/ml [
33]. The IC50 of propofol as single drug is 10 μM (1.78 μg/ml) and the dose of propofol used for combination therapy is 5 μM (0.89 μg/ml) (Fig.
1), suggesting that the effective concentrations of propofol in CML are clinically achievable. Importantly, our results obtained from CML xenograft mouse model further demonstrate the in vivo efficacy of propofol and its synergistic effects with BCR-ABL TKIs (Fig.
4). It has also been shown that propofol enhances paclitaxel- or cisplatin-induced apoptosis in ovarian and cervical cancer cells [
13,
17]. Our results together with the previous studies demonstrate that propofol is a potential candidate for combination therapy in cancer treatment.
CML is recognized to be a hematopoietic stem cell disorder and CD34 cells serve as a reservoir for disease relapse [
8]. In line with cell lines results, propofol also effectively induces apoptosis and inhibits colony formation of CD34 cells derived from 15 CML patients (Fig.
2a and
b). Targeting tumor cells while sparing normal counterparts is critical for targeted therapy. Compared to CML CD34 cells, propofol is less effective in targeting NBM CD34 cells (Fig.
2a and b) and combination of propofol with dasatinib selectively targets CML but not NBM CD34 cells (Fig.
2c and d). The inhibitory effects of propofol in cancer are mostly demonstrated by cancer cell lines, we are the first to show propofol’s therapeutic effects in patient primary stem/progenitor cells.
We further investigated the molecular mechanism of propofol’s action in CML. It dose-dependently decreases phosphorylation levels of Akt at S473 and T308, mTOR at S2448 and S2481 in CML cells (Fig.
3a). Consistently, propofol decreases the phosphorylation of downstream effectors of mTOR pathway including S6 and 4EBP1 (Fig.
3a). Overexpression of constitutively active Akt significantly abolishes propofol’s effects in CML cells (Fig.
3c and
d), confirming that propofol acts on CML via suppression Akt/mTOR pathway. Interestingly, we also find that short time exposure of CML cells to imatinib results in decreased levels of p-Akt, p-mTOR and p-S6. Further decreased phosphorylation levels of these molecules are observed in cells in the presence of both imatinib and propofol (Fig.
3b). These results suggest that propofol augments TKI’s effect by its combinatory effects in further decreasing Akt/mTOR pathway. Synergism between imatinib and inhibitors of PI3K, Akt or mTOR have been observed in CML cells [
34]. In line with previous work, we demonstrate that combination of rapamycin (mTOR inhibitor) and imatinib was significantly more effective than single drug alone in inhibiting proliferation and inducing apoptosis in multiple CML cell lines (Additional file
1: Figure S2). These suggest that propofol acts similarly as mTOR inhibitors in CML. In addition, propofol has advantages over these inhibitors due to the fact that propofol has already been used in clinics.