Pterostilbene downregulates BCR/ABL and induces apoptosis of T315I-mutated BCR/ABL-positive leukemic cells

In this study, we examined the antileukemic effects of pterostilbene, a natural methylated polyphenol analog of resveratrol that is predominantly found in berries and nuts, using various human and murine leukemic cells, as well as bone marrow samples obtained from patients with leukemia. Pterostilbene administration significantly induced apoptosis of leukemic cells, but not of non-malignant hematopoietic stem/progenitor cells. Interestingly, pterostilbene was highly effective in inducing apoptosis of leukemic cells harboring the BCR/ABL fusion gene, including ABL tyrosine kinase inhibitor (TKI)-resistant cells with the T315I mutation. In BCR/ABL+ leukemic cells, pterostilbene decreased the BCR/ABL fusion protein levels and suppressed AKT and NF-κB activation. We further demonstrated that pterostilbene along with U0126, an inhibitor of the MEK/ERK signaling pathway, synergistically induced apoptosis of BCR/ABL+ cells. Our results further suggest that pterostilbene-promoted downregulation of BCR/ABL involves caspase activation triggered by proteasome inhibition-induced endoplasmic reticulum stress. Moreover, oral administration of pterostilbene significantly suppressed tumor growth in mice transplanted with BCR/ABL+ leukemic cells. Taken together, these results suggest that pterostilbene may hold potential for the treatment of BCR/ABL+ leukemia, in particular for those showing ABL-dependent TKI resistance.

www.nature.com/scientificreports/ in several fields of human health, as confirmed by the number of publications concerning its antioxidative and anti-inflammatory activities. Given its functional activities, resveratrol is also quite popular as a nutritional supplement 7,8 . Recent reports have further demonstrated the antiproliferative and proapoptotic activities of resveratrol against various human cancers, including leukemia 7,9 . However, the low bioavailability of the parental compound, as consequence of its poor resorption and extensive biotransformation, is a limitation for the therapeutic use of this molecule 10 . Similar to resveratrol, pterostilbene (trans-3,5-dimethoxy-4-hydroxystilbene), a natural dimethyl ether analog of resveratrol, is predominantly found in berries and nuts. From a structural point of view, pterostilbene and resveratrol are quite similar 11,12 ; however, pterostilbene has superior oral adsorption and metabolic stability owing to the presence of only one hydroxy group in its structural backbone 6 . Furthermore, pterostilbene has been reported to exhibit anticancer activities via various mechanisms in several common malignant tumors, including hematologic malignancies [13][14][15][16][17][18][19] .
Herein, we explored the therapeutic potential of pterostilbene on various hematological malignancies, especially in BCR/ABL + leukemia, and also explored the underlying molecular and cellular mechanism by which pterostilbene exerts its effects on these leukemic cells.

Results
Pterostilbene specifically induces apoptosis of BCR/ABL + leukemic cells, including TKI-resistant T315I-mutated BCR/ABL + cells. Firstly, we assessed the apoptosis-inducible activity of pterostilbene in vitro. The addition of pterostilbene significantly induced apoptosis of various human leukemic cells, but not of human umbilical cord blood-derived CD34 + normal hematopoietic stem/progenitor cells and murine hematopoietic cells (32D cells; Fig. 1a, Supplementary Figs. 1 and 2). Interestingly, pterostilbene strongly induced apoptosis of BCR/ABL + leukemic cells than of BCR/ABL − leukemic cells (Fig. 1a). In contrast to 32D-p210 BCR/ ABL cells, T315I-32D-p210 BCR/ABL cells were resistance to dasatinib; nonetheless, pterostilbene was still able to induce apoptosis of most of these cells (Fig. 1b). We further assessed the apoptosis-inducible effect of pterostilbene using frozen bone marrow mononuclear cells (BMMNCs) isolated from four patients: three with CML in the chronic phase at diagnosis and one with relapsed ALL harboring the T315I ABL mutation. Pterostilbene administration also significantly induced apoptosis of BMMNCs from all four patients (Fig. 1c).
Pterostilbene synergizes with a MEK/ERK inhibitor to induce apoptosis of BCR/ABL + cells. We further observed the effect of pterostilbene administration on the MEK/ERK pathway, which is also an important target/effector of BCR/ABL along with AKT/NF-κB and is associated with pro-survival events [22][23][24][25][26][27] . Interestingly, we found that pterostilbene administration enhanced ERK activation in K562 and T315I-32D-p210 BCR/ABL cells (Fig. 3a, Supplementary Fig. 5). In contrast, the MEK inhibitor U0126 inhibited ERK activation but did not enhance caspase activity or induce apoptosis in either cell line (Fig. 3a, Supplementary Fig. 5). However, the combined administration of pterostilbene and U0126 inhibited ERK activation and synergistically induced caspase activation and apoptosis in both cell lines ( Fig. 3a,b, Supplementary Fig. 5).
Pterostilbene-induced BCR/ABL downregulation results from caspase-dependent cleavage. Caspase-dependent cleavage is involved in the decrease of BCR/ABL levels in BCR/ABL + leukemic cells triggered by the administration of several antileukemic drugs 26,28-32 ; thus, we examined whether caspase activation could also be associated with the observed pterostilbene-induced BCR/ABL downregulation. We incubated K562 and T315I-32D-p210 BCR/ABL cells with pterostilbene in the absence or presence of a pan-caspase inhibitor, z-VAD. Pterostilbene administration induced the activation of caspase-3 and caspase-9, and reduced BCR/ABL levels in both cell lines; however, presence of z-VAD inhibited these events, suggesting that BCR/ABL downregulation due to pterostilbene administration primarily reflects caspase-mediated protein degradation (Fig. 4a, Supplementary Fig. 6). We investigated further the effect of z-VAD on the apoptotic effect of pterostilbene in BCR/ABL + cells and found that addition of z-VAD markedly, but not completely, inhibited the pterostilbeneinduced apoptosis (Fig. 4b).
While many studies have recently found that the ubiquitin-proteasome system plays a key role in endoplasmic reticulum (ER) stress 33 , caspase activation caused by proteasome inhibition-induced ER stress has been suggested by several reports to contribute to BCR/ABL downregulation 28,32 . Therefore, we investigated whether pterostilbene could have a proteasome-inhibitory effect. When T315I-32D-p210 BCR/ABL cells were treated with pterostilbene, ubiquitinated protein levels increased over time, suggesting proteasome inhibition, followed by the activation of caspases and downregulation of BCR/ABL (Fig. 4c, Supplementary Fig. 7). Furthermore, in T315I-32D-p210 BCR/ABL cells treated with pterostilbene, ER stress sensors 34 , such as the protein kinase R-like endoplasmic reticulum kinase (PERK) and the activating transcription factor 6 (ATF6), were activated (Fig. 4d, Supplementary Fig. 8). In addition, pterostilbene treatment also promoted the expression of the CCAAT/  Fig. 4d, Supplementary Fig. 8), which was induced by both the PERK/eIF2a signaling pathway and the cleaved activated form of ATF6 (ATFp50), and consequently activated the caspase cascade [34][35][36] . Together, these results suggest that the downregulation of BCR/ABL due to pterostilbene administration involves caspase activation downstream of proteasome inhibition-induced ER stress.

Pterostilbene administration effectively represses the proliferation of BCR/ABL + leukemic cells in vivo.
Lastly, we examined the effect of pterostilbene administration on the proliferation of K562 and www.nature.com/scientificreports/ T315I-32D-p210 BCR/ABL leukemic cells in vivo. We transplanted the leukemic cells subcutaneously into the right flank of immunodeficient mice and orally administered the investigational compounds. In mice transplanted with K562 cells that stably expressed luciferase (Luc-K562 cells), pterostilbene and dasatinib administration significantly inhibited tumor growth as compared with in the vehicle control (Fig. 5a,b). In mice transplanted with T315I-32D-p210 BCR/ABL cells, although dasatinib administration exhibited no inhibitory effect on tumor growth, pterostilbene administration significantly prevented tumor growth (Fig. 5c,d). General toxicities of the reagents were also measured, and no noticeable behavioral changes or morbid consumption, such as significant weight loss or death, were observed (data not shown).

Discussion
Phytochemicals are natural compounds from plants that represent vital resources for cancer treatment 37-41 , including taxol analogs, vinca alkaloids, and podophyllotoxin analogs 41 . These phytochemicals have significant antitumor potential, but adverse events due to toxicity are often a challenge. In the present study, we found that pterostilbene, a natural polyphenolic compound and a methylated analog of resveratrol, significantly induces apoptosis in BCR/ABL + cells, including of dasatinib-resistant cells harboring the T315I ABL mutation. In most in vitro experiments, we used 200 μM pterostilbene, which is a higher concentration than that used in previous reports in which the antileukemic effect of pterostilbene was observed (~ 150 μM) [15][16][17][18] ; nonetheless, no apparent negative effect was herein observed on normal hematopoietic stem/progenitor cells regarding their survival. We have also confirmed that even lower concentrations of pterostilbene can effectively induced apoptosis of BCR/ ABL + cells with longer incubation time ( Supplementary Fig. 2). www.nature.com/scientificreports/ Pterostilbene was also shown to significantly reduced the levels of BCR/ABL and inhibited its downstream targets AKT/NF-κ-B. However, pterostilbene administration enhanced the activation of ERK, which is a critical target/effector of BCR/ABL and is associated with antiapoptotic actions [22][23][24][25][26][27] . These findings agree with a previous study that showed that downregulation of BCR/ABL activates MEK/ERK via prosurvival growth factormediated signals through a BCR/ABL-mediated negative feedback 27 . Importantly, pterostilbene along with a MEK inhibitor were shown to synergistically enhance caspase activation and consequently trigger apoptosis of BCR/ABL + leukemic cells.
Our results suggest that pterostilbene-promoted downregulation of BCR/ABL involves caspase activation triggered by proteasome inhibition-induced ER stress. It has been reported that BCR/ABL is associated with an increase in proteasome activity 42 and that proteasome activity is higher in BMMNCs from patients with CML than in healthy controls 43 , suggesting that CML cells may be more susceptible to proteasome inhibition. In www.nature.com/scientificreports/ fact, it has been reported that BCR/ABL + cells are more sensitive than BCR/ABLcells to induced apoptosis by proteasome inhibitors 42,44 . It was also suggested that inhibition of the proteasome to trigger ER stress could be an alternative strategy for treating CML 44 . Therefore, we speculate that the mechanism by which pterostilbene strongly induces BCR/ABL + cells to undergo apoptosis is also largely due to caspase activation as a consequence of ER stress caused by proteasome inhibition. Indeed, in this study, the pan-caspase inhibitor z-VAD significantly, but not completely, prevented the pro-apoptotic effects of pterostilbene, but it also induced apoptosis in BCR/ ABL − leukemic cells, suggesting that other mechanisms are involved in the pterostilbene downstream effects, as previously reported [15][16][17][18][19] . Although many studies have reported that resveratrol exerts antileukemic effects 45 , opposite results were obtained in vivo, which may be due to its low bioavailability and because it mainly exists in vivo as glucuronideconjugated and sulfated metabolites [46][47][48] . In contrast, pterostilbene exhibits superior oral adsorption and metabolic stability compared with resveratrol 6,10 . Herein, pterostilbene was orally administered to mice transplanted with K562 or T315I-mutated BCR/ABL + leukemic cells. We found that pterostilbene administration significantly inhibited leukemic cell proliferation at a dose that did not cause general toxicity in vivo. Oral administration of pterostilbene has also been reported to be safe and well-tolerated in randomized, double-blind, placebocontrolled studies in healthy subjects or patients with hypercholesterolemia 49,50 .
Collectively, pterostilbene administration may be a novel noninvasive treatment option for patients with BCR/ABL + leukemia, especially for those resistant or intolerant to TKIs.

Materials and methods
Cells. The study was conducted in accordance with the principles of the Declaration of Helsinki and was approved by the Ethics Committee of the Tokai University School of Medicine (number 20I-34). Written informed consent was obtained from all patients with BCR/ABL + leukemia and umbilical cord blood (CB) donors prior to sample collection. BMMNCs were isolated from the samples using Lymphoprep (StemCell Technologies) density gradient centrifugation. The CB-CD34 + cell fraction was prepared using the CD34 Progenitor Cell Isolation Kit (Miltenyi Biotec). BMMNCs from patients with leukemia and CB-CD34 + cells from healthy donors were frozen in a medium supplemented with dimethyl sulfoxide (DMSO) and fetal calf serum using a step-down freezing procedure, and stored in liquid nitrogen. Aliquots of frozen samples were thawed immediately before use.  www.nature.com/scientificreports/ Center, Hayashibara Biochemical Laboratories. K562 cells stably expressing luciferase (Luc-K562 cells) were obtained by transduction of lentivirus containing the luciferase-enhanced green fluorescent protein gene (CSII-CMV-Luciferase2-EGFP). The 32D-p210 BCR/ABL and T315I-32D-p210 BCR/ABL cell lines were established by infection of 32D cells with MSCV-BCR-ABL-ires-GFP and MSCV-BCR-ABL1-T315I mutant-ires-GFP, respectively, as previously described 51 . The cells were maintained in RPMI 1640 medium supplemented with 10% (20% for KCL22 cells) heat-inactivated fetal bovine serum and antibiotics (100 U penicillin/mL and 100 μg streptomycin/ mL) at 37 °C in a humidified 5% CO 2 atmosphere.
Reagents. Pterostilbene was kindly provided by the Molecular Physiological Chemistry Laboratory (Tokyo, Japan), dissolved in DMSO to yield a 100 mM stock solution, and stored at − 20 °C. The TKI dasatinib, the pan-caspase inhibitor z-VAD, and the MEK inhibitor U0126 were purchased from Cayman Chemical, Peptide Institute, and Selleck Chemicals, respectively.
Cell culture. Reagents were added at the indicated concentrations to 96-well culture plates containing 1 × 10 4 cells/well. Unless otherwise specified, the concentrations of pterostilbene, dasatinib, z-VAD, and U0126 were 200 μM, 20 nM, 30 mM, and 10 μM, respectively. The reagents in the treatment groups were diluted with RPMI-1640, and the corresponding volumes of DMSO and RMPI-1640 were added to the vehicle control group. When pterostilbene was added to the cell culture media along with z-VAD or U0126, pterostilbene was added 2 or 3 h after addition of the other inhibitors. Unless otherwise specified, 20 h after the addition of pterostilbene, the cells were harvested. Capillary western immunoassay. Cell pellets were suspended in 0.1 mL of ice-cold RIPA buffer (Invitrogen) and incubated on ice for 1 h. Protein concentrations were determined using a DC protein assay (Bio-Rad). Protein expression was evaluated by capillary western immunoassay using a Wes machine (ProteinSimple), according to the manufacturer's instructions. This system automates protein loading, separation, immunoprobing, washing, and detection, and allows absolute protein quantitation. Briefly, samples were mixed with Simple Western Sample Buffer and standards to a final concentration of 1 μg/μL, and were then reduced and denatured. The prepared samples, primary and secondary antibodies, and chemiluminescent substrate were dispensed in microliter volumes into the designated wells in an assay plate. The prepared assay plate and a capillary cartridge were loaded into the Wes machine, and the solutions were run through the capillaries. Proteins were separated in the capillaries as they migrated through a stacking and separation matrix. The separated proteins were immobilized on the capillary wall via a proprietary, photoactivated capture chemical reaction. Target proteins were then identified with a primary antibody, and subsequent immunodetection using a horseradish peroxidaseconjugated secondary antibody and chemiluminescent substrate. The chemiluminescence reactions were measured and digital blot images were captured. Antibodies against c-ABL, phospho-c-ABL, ABL, phospho-AKT, NF-κB p65, phospho-NF-κB p65, caspase-3, cleaved caspase-3, caspase-9, cleaved caspase-9, ubiquitin, p27, ERK, phospho-ERK, PERK, phospho-PERK, and phospho-eIF2α were purchased from Cell Signaling Technology. Antibodies against eIF2α and CHOP were purchased from Santa Cruz Biotechnology. Antibodies against ATF6 and ATF6 p50 were purchased from Novus Biologicals. The antibody against glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was purchased from Sigma-Aldrich.

Measurement of apoptosis.
Reverse transcription quantitative polymerase chain reaction (PCR). RNA was isolated using the RNeasy Mini Kit (Qiagen) and was reverse transcribed. Each target cDNA was amplified via PCR on the same plate using the TaqMan Gene Expression Assays (Thermo Fisher Scientific) and the ABI 7300 Real-Time PCR System (Thermo Fisher Scientific). The TaqMan probe used was derived from BCR/ABL (Thermo Fisher Scientific; Assay ID: Hs03024541). The relative amounts of the target genes were determined with reference to 18S rRNA. Comparative threshold cycle (C T ) analysis was used to quantify transcripts. The values were calculated using the 2 −ΔΔCT method.
Transplantation and treatment procedures. Eight-week-old female immunodeficient SHO mice were purchased from CLEA Japan. A mixture of 2 × 10 6 Luc-K562 cells or T315I-32D-p210 BCR/ABL cells and basement membrane matrix (BD Biosciences) was subcutaneously injected into the right flank of the mice. Day 8 after transplantation, the mice were administered vehicle, dasatinib (10 mg/kg/day), or pterostilbene (25 mg/kg/day) orally for a total of 14 times in 18 days. The tumor volume was measured with calipers at the times indicated in Fig. 5a,c. The general toxicity of the reagents was measured during the experiments. All experimental procedures and protocols involving animals were reviewed and approved by the Animal Care Committee of the Tokai University and were in compliance with the ARRIVE guidelines 52,53 , and all methods were carried out in accordance with relevant guidelines and regulations.
Bioluminescence imaging. Bioluminescence imaging was performed with a highly sensitive, cooled CCD camera mounted in a light-tight specimen box (in vivo imaging system [IVIS]; Xenogen Corporation), as described previously 54  www.nature.com/scientificreports/ D-luciferin (150 mg/kg). The light emitted from the mice was detected using the IVIS camera, integrated, digitized, and displayed. The total flux of photons on the images, which correlated well with tumor volume, was estimated by region of interest (ROI) measurements, which converted the surface radiance (photons/s/cm 2 /sr) to the total flux of photons (photons/s) using the Living Image Software (Caliper Life Sciences).

Statistics.
All experimental results were expressed as the arithmetic mean and standard deviation values.
Student's t-test was used to evaluate the statistical significance of the differences between unpaired groups. Statistical significance was set at P < 0.05. www.nature.com/scientificreports/ Publisher's note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

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