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The International Journal of Biochemistry & Cell Biology 65 (2015) 1–11

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The International Journal of Biochemistry
& Cell Biology
journal homepage: www.elsevier.com/locate/biocel

Curcumin potentiates the anti-leukemia effects of imatinib by
downregulation of the AKT/mTOR pathway and BCR/ABL gene
expression in Ph+ acute lymphoblastic leukemia
Yong Guo a,1 , Yi Li b,1 , Qingqing Shan a , Guangcui He a , Juan Lin a , Yuping Gong a,∗
a
Department of Hematology, West China Hospital of Sichuan University, China
b
Department of Human Sciences, Texas A&M University-Kingsville, Kingsville, TX 78363, USA

a r t i c l e i n f o a b s t r a c t

Article history: Philadelphia chromosome-positive acute lymphoblastic leukemia (Ph+ ALL) is triggered by BCR/ABL
Received 14 November 2014 and SRC family tyrosine kinases. They interact with each other and subsequently activate downstream
Received in revised form 24 March 2015 growth-signaling pathways, including Raf/MEK/ERK, Akt/mTOR, and STAT5 pathways. Although imatinib
Accepted 1 May 2015
is the standard treatment for Ph+ leukemia, response rate of Ph+ ALL to imatinib is low, relapse is frequent
Available online 12 May 2015
and quick. Studies have documented the potential anti-tumor activities of curcumin. However, whether
curcumin can be used in the therapy for Ph+ ALL remains obscure. Here, we reported that curcumin
Keywords:
induced apoptosis by inhibition of AKT/mTOR and ABL/STAT5 signaling, down-regulation of BCR/ABL
Philadelphia chromosome-positive acute
lymphoblastic leukemia
expression, and induction of the BCL2/BAX imbalance. Curcumin exerted synergetic anti-leukemia effects
Curcumin with imatinib by inhibition of the imatinib-mediated overactivation of AKT/mTOR signaling and down-
Imatinib regulation of BCR/ABL gene expression. In primary samples from Ph+ ALL patients, curcumin inhibited
Anti-leukemia effect cellular proliferation and down-regulated constitutive activation of growth-signaling pathways not only
AKT/mTOR in newly diagnosed patients but also in imatinib-resistant patients. In Ph+ ALL mouse models, curcumin
exhibited synergetic anti-leukemia effects with imatinib. These results demonstrated that curcumin
might be a promising agent for Ph+ ALL patients.
© 2015 Published by Elsevier Ltd.

1. Introduction include AKT/mTOR, RAF/MEK/ERK and STAT5, which are involved in
controlling the proliferation, differentiation and apoptosis of lym-
The Philadelphia (Ph) chromosome is the most frequent genetic phoid precursors (Sawyers, 1997; Skorski et al., 1997; Meyn et al.,
aberration in acute lymphoblastic leukemia (ALL), particularly in 2006). In the pre-imatinib era, the Ph+ ALL prognosis was poor, with
adult ALL patients (Piccaluga et al., 2006). It is associated with a poor five-year overall survival rates of 10–20% (Jones and Saha, 2005;
prognosis (Pui and Evans, 2006; Moorman et al., 2007). The recipro- Dombret et al., 2002). The tyrosine kinase inhibitor (TKI), imatinib,
cal translocation, t(9;22), generates a BCR/ABL fusion gene encoding down-regulates BCR/ABL activity and is widely used to clinically
the BCR/ABL fusion protein, which has constitutively active tyrosine treat Ph+ leukemia (Hochhaus et al., 2009; Gruber et al., 2009;
kinase activity (Faderl et al., 2003). Although, the BCR/ABL protein Schultz et al., 2009). However, the response of Ph+ ALL to imatinib
kinase is the primary cause of Ph+ ALL, SRC kinases are also sine qua monotherapy is low, and the response duration is short. Therefore,
non factors that contribute to the disease (Lugo et al., 1990; Hu et al., resistance and relapse are continuing problems (Druker et al., 2001;
2004). BCR/ABL and SRC kinases interact with one another, then Ottmann et al., 2002). Many studies have indicated that imatinib
activates the downstream signaling pathways. These pathways resistance, including primary and acquired resistance, is the main
reason for the poor response of Ph+ ALL to imatinib monotherapy.
Many mechanisms are involved in imatinib resistance. Generally,
there are two types of resistance mechanisms: BCR/ABL-dependent
∗ Corresponding author at: Department of Hematology and State Key Lab- and BCR/ABL-independent resistance. Mutations in the BCR/ABL
oratory of Biotherapy, West China Hospital of Sichuan University, 37# Guo kinase domain (KD) are the most prevalent BCR/ABL-dependent
Xue Xiang, Chengdu, Sichuan Province 610041, China. Tel.: +86 28 85422370;
resistance mechanisms (Bixby and Talpaz, 2009; Soverini et al.,
fax: +86 28 85423921.
E-mail address: gongyuping94@aliyun.com (Y. Gong). 2006). BCR/ABL-independent resistance involves alterations in the
1
Co-first authors. dynamics of drug import and efflux, and other kinases or parallel

http://dx.doi.org/10.1016/j.biocel.2015.05.003
1357-2725/© 2015 Published by Elsevier Ltd.
2 Y. Guo et al. / The International Journal of Biochemistry & Cell Biology 65 (2015) 1–11

signaling pathways that sustain leukemic-cell survival (Burchert Biotech.CO., Ltd. 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyl-2H-
et al., 2005; Wu et al., 2008; Suzuki et al., 2010). These two mecha- tetrazolium bromide (MTT) was obtained from Sigma. IMDM
nisms may synergize to prevent leukemia eradication. In our previ- medium, penicillin/streptomycin and fetal bovine serum (FBS)
ous experiments, we discovered that the apoptosis rate in Ph+ ALL were obtained from Hyclo Company.
cells remained low after 24 h of exposure to imatinib, which indi-
cated that imatinib did not immediately induce apoptosis. Further 2.2. Cell Lines, patient specimens and mice
studies have shown that imatinib treatment increased the activa-
tions of AKT/mTOR signaling. Additionally, imatinib had little effect SUP-B15, a human Ph+ B-ALL cell line expressing P190-BCR/ABL,
on the activation of LYN kinase, one of SRC kinases (Guo et al., 2012). was obtained from American Type Culture Collection (ATCC)
Thus, we hypothesized that aberrant signaling from the AKT/mTOR (Rockville, MD). The human monoblastic leukemia cell line THP-
and LYN kinase pathways may account for the poor response of Ph+ 1 and human acute T-lymphoblastic leukemia cell line CEM
ALL to imatinib, at least partially, and that they may be the under- were obtained from ATCC and cultured in RPMI-1640 medium
lying basis of imatinib resistance. Methods to prevent or overcome supplemented with 10% FBS, 100 U/mL penicillin and 100 g/mL
imatinib resistance in Ph+ ALL remain an active area of research. streptomycin and in 5% CO2 incubator at 37 ◦ C. Primary leukemia
Second-generation tyrosine kinase inhibitors, such as nilotinib and samples were obtained from five Ph+ ALL patients diagnosed at
dasatinib, can overcome imatinib resistance to some extent. While the Department of Hematology in the West China Hospital of
patients treated with these drugs often gain complete remission Sichuan University. Written informed consent was obtained from
(CR) quickly, with CR rates of approximately 90%. However, the the patients, and the study was approved by the local ethics
duration of CR is often short (Redaelli et al., 2009; Ottmann et al., committee. Normal human peripheral blood mononuclear cells
2007). As above-mentioned reports have indicated, targeting a (PBMCs) were obtained from the blood of donors. The mononuclear
complex disease such as Ph+ ALL with a single agent will eventually cells were isolated by density centrifugation (Ficoll-Hypaque). The
induce drug resistance. Therefore, preventing TKI resistance is the cells were then cultured in IMDM medium supplemented with 10%
goal of achieving durable responses. Combining TKIs with steroids, FBS, 100 U/mL penicillin and 100 g/mL streptomycin in a 5% CO2
chemotherapy or a-interferon has shown some efficacy (Vignetti incubator at 37 ◦ C.
et al., 2007; Boulos et al., 2011; Wassmann et al., 2003; Piccaluga All female BALB/c null mice used in this study were bred in a spe-
et al., 2008). However, these treatments were accompanied by side cific pathogen-free environment in the experimental animal center
effects that decreased their tolerability to patients. Therefore, there of West China Hospital, Sichuan University. All animal procedures
is an urgent need to develop new therapeutic strategies. were approved by the Institutional Animal Care and Use Commit-
Curcumin, a yellow polyphenol, is the principle active com- tee of the Sichuan University West China Hospital, following the
pound of the perennial herb, Curcuma longa. Extensive research guideline of the US National Institutes of Health.
within the last decade has revealed that curcumin is a promis-
ing, pharmacologically safe anti-tumor agent that functions as a
2.3. MTT assay
chemosensitizer, a radiosensitizer, and a multi-targeted inhibitor
to signaling proteins, particularly the mTOR protein (Goel and
The anti-proliferative effects of curcumin on SUP-B15 cells,
Aggarwal, 2010; Kunnumakkara et al., 2008). Considering the
CEM cells, THP-1 cells and Ph+ ALL primary cells were deter-
prominent effects of curcumin on multiple protein kinases, we
mined with an MTT assay. Briefly, the cells were incubated for
hypothesized that curcumin may sensitize Ph+ ALL cells to imati-
the indicated time at 37 ◦ C in triplicate in a 96-well plate in
nib by deregulating multiple protein kinases, thus potentiating the
the presence or absence of the indicated drugs in a final vol-
efficiency of imatinib and subsequently reducing the incidence of
ume of 100 ␮L. Thereafter, 20 ␮L of MTT solution, of concentration
imatinib resistance. Based on this hypothesis, we investigated the
5 mg/mL in PBS, was added to each well. After 4 h of incu-
anti-leukemia effects of curcumin alone or in combination with
bation at 37 ◦ C, 100 ␮L of an SDS-isobutanol-HCl solution was
imatinib on Ph+ ALL. We have identified curcumin as a potent
added, and incubation was continued overnight at room tem-
growth inhibitor and a sensitizer of imatinib both in vitro and in vivo.
perature. Subsequently, the optical density (OD) was measured
Furthermore, we demonstrated that curcumin potentiates the effi-
using ␮Quant MQX200 Microplate Spectrophotometer (Biotek) at
ciency of imatinib by combating the activation of the AKT/mTOR
a wavelength of 570 nm. The cell viability was displayed as a
signaling mediated by imatinib and by down-regulating the expres-
percentage according to the following formula: OD(experiment
sion of the BCR/ABL gene.
samples) − OD(blank)/[OD(control) − OD(blank)] × 100%. The per-
centage of cell growth inhibition was calculated as follows:
Cell growth inhibition (%) = OD(control) − OD(experiment sam-
2. Materials and methods
ples)/[OD(control) − OD(blank)] × 100%. Synergistic cytotoxicity
was determined by calculating the interaction index (I) according
2.1. Reagents
to the classic isobologram equation as follows (Berenbaum, 1989):
I = (D)1/(Dx)1 + (D)2/(Dx)2, where Dx is the concentration of com-
Curcumin was purchased from Sigma. The 100 mM
pound required to produce the effect, in this case 50% inhibition
stock solution of curcumin in DMSO and the 10 mM stock
of cell growth, alone, and (D)1 and (D)2 are the concentration of
solution of imatinib (Novartis) in DMSO were stored at
both compounds that produce the same effect. It indicates an addi-
−20 ◦ C. The phosphospecific antibodies against ABL(Tyr245),
tive effect when I = 1, a synergic effect when I < 1, and it means
PDK1(Ser241), PTEN(Ser380), AKT(ser473), GSK3␤(ser9),
antagonism between two drugs when I > 1.
mTOR(ser2448), 4EBP1(Thr37/46), P70S6(Thr389), cRAF(ser338),
MEK1/2(ser217/221), ERK1/2(Thr202/Tyr204) and the antibodies
against AKT, mTOR, 4EBP1, P70S6, MEK1/2, LC3, BAX, BCL2 were 2.4. Light microscopy
obtained from Cell Signaling Technologies. The phosphospecific
antibodies against LYN(Tyr396) and STAT5(Tyr694) were pur- SUP-B15 cells were seeded at a density of 2 × 106 cells in each
chased from Abcam. The monoclonal antibody to human CD19 well of a 6-well culture plate and cultured with the indicated drugs
was purchased from Gene Tech Company Limited (Shanghai). The for 24 h. The changes in cellular morphology were observed using
Annexin V-FITC apoptosis detection kit was obtained from KeyGen a light microscope (Olympus).
Y. Guo et al. / The International Journal of Biochemistry & Cell Biology 65 (2015) 1–11 3

2.5. Western blot analysis and 5 ␮L of PI was added after 10 minutes. Next, the cells were incu-
bated at room temperature for 15 min in the dark. Then, 400 ␮L of
Whole cell extracts were prepared in RIPA lysis buffer and 80 ␮g binding buffer was added, and the stained cells were analyzed using
protein extracts were quantitated and loaded onto a 6–15% sodium a Cytomics FC500 flow cytometer equipped with CXP software. For
dodecyl sulfate–polyacrylamide gel. After electrophoresis, the pro- each analysis, 10,000 events were recorded.
teins were electro-transferred to a nitrocellulose membrane. The
membrane was incubated with primary antibody, washed and
incubated with horseradish peroxidase (HRP)-conjugated sec-
2.7. Analysis of the expression of BCR/ABL gene at mRNA level
ondary antibody, and finally, the signals were detected using
the enhanced chemiluminescence (ECL) detection system and
SUP-15 cells were treated with the indicated drugs for 24 h. Then
film (Bio-Rad Laboratories Inc.), according to the manufacturer’s
total RNA was isolated and 2 ␮g RNA was converted to cDNA by
instructions.
SuperScript reverse transcriptase. Changes in BCR/ABL gene expres-
sion were analyzed using semi-quantitative RT-PCR, with ␤-actin
2.6. Apoptosis analysis as an internal control. The primer sequences for BCR/ABL gene were
5 -CCG GAG TTT TGA GGA TTG CGG A3 (sense) and 5 -TTG GAG TTC
The cells were treated with the indicated drugs for 24 h, and CAA CGA GCG GC3 (anti-sense). The PCR products (10 ␮L) were
2 × 105 cells were harvested, washed with PBS and resuspended in analyzed by electrophoresis on a 1.5% (w/v) agarose gel, photo-
100 ␮L of binding buffer. Then, 5 ␮L of annexin V-FITC was added, graphed, and quantified by densitometric scanning.

Fig. 1. Cytotoxic effects of curcumin alone or in combination with imatinib on SUP-B15 cells. Same number of SUP-B15 cells (A), CEM cells (B), and THP-1 cells (C) were
incubated with the indicated concentration of curcumin for 1 day, 3 days and 5 days. At the end of incubation, the cell survival rates were determined by MTT assay. The cells
were incubated with imatinib alone or in combination with 5 ␮M, 10 ␮M, 15 ␮M and 20 ␮M curcumin for 72 h. At the end of incubation, the cell viability (D) and the IC50
of imatinib (E) were determined by MTT assay. After 24 h of treatment with curcumin and imatinib (alone or combined), morphological changes of the SUP-B15 cell were
observed with a light microscope (E ×400) (F). Data were obtained from three independent experiments, and * indicates p < 0.05 compared with the group of imatinib alone.
4 Y. Guo et al. / The International Journal of Biochemistry & Cell Biology 65 (2015) 1–11

2.8. In vivo studies The spleens were also collected and were fixed in 10% formalin to
examine the expression of human CD19 by immunohistochemistry
Female BALB/c null mice aged 5–6 weeks old were injected assay using mouse anti-human CD19.
intraperitoneally with 2 mg of cyclophosphamide per mouse on day
2 and day 3. The mice were irradiated at a dose of 400 cGy on day −1
and then intravenously injected with 107 SUP-B15 cells via tail vein 2.9. Statistical analysis
on day 0. After 7 days, the mice were injected intraperitoneally with
50 mg/kg day curcumin, 5 mg/kg day imatinib, or 25 mg/kg day cur- The IC50 , cell viability, and apoptosis percent were analyzed
cumin plus 5 mg/kg day imatinib over a 14-day period. During this with a one-way ANOVA and independent sample t-test. P values
period, the mice were injected once daily for 5 days per week. The <0.05 were considered statistically significant. All of the statis-
mice were then killed, and bone marrow samples were obtained to tical analyses were performed using the software SPSS 16.0 for
detect the BCR/ABL gene expression changes using RT-PCR methods. Windows.

Fig. 2. Curcumin induced significantly more apoptotic events of SUP-B15 cells compared with normal human PBMCs. (A) SUP-B15 cells were treated with the indicated
concentration of curcumin and imatinib alone or in combination for 24 h. The percent of apoptotic cells was examined by flow cytometry using the Annexin V-FITC/PI
apoptosis detection kit. Data were obtained from three independent experiments and expressed as mean ± SE. * represents significant difference (p < 0.05), N.S represents
no significant difference. (B) In non-BCR/ABL positive leukemia cell line CEM and THP-1 cells, curcumin induces leukemia cell apoptosis, while in normal human PBMCs,
curcumin-induced apoptosis was few.
Y. Guo et al. / The International Journal of Biochemistry & Cell Biology 65 (2015) 1–11 5

3. Results Because 15 ␮M curcumin combined with imatinib had strongest
synergistic effect and their interaction index was the lowest, which
3.1. Curcumin significantly induces cytotoxic effects and was only 0.64, therefore, the concentration of curcumin was used
potentiates the anti-leukemia effects of imatinib in SUP-B15 cells as 15 ␮M in the later studies when curcumin and imatinib were
compared with normal human PBMCs combined.
Additionally, to confirm whether the anti-leukemia role of cur-
First, we examined the cytotoxic effects of curcumin on cumin was cell- dependent or not, we also examined the effects
SUP-B15 cells. Using an MTT assay, we found that curcumin of curcumin on non-BCR/ABL positive leukemia cell line CEM and
inhibited the proliferation of SUP-B15 cells in a dose- and THP-1 cells, and normal human PBMCs. Our results showed that
time-dependent manner (Fig. 1A). The IC50 of curcumin at 72 h curcumin inhibited the proliferation of CEM cells (Fig. 1B) and
was 30.59 ± 7.06 ␮M. The IC50 of imatinib alone at 72 h was THP-1 cells (Fig. 1C) in a time- and dose-dependent manner, with
1.24 ± 0.48 ␮M, and the cell viability was 60.73 ± 3.40% after the IC50 at 72 h was 32.78 ± 5.32 ␮M, 27.13 ± 1.49 ␮M respectively.
1 ␮M of imatinib for 72 h. When dosed in combination with Curcumin had minimal cytotoxic effects on normal human PBMCs
5 ␮M, 10 ␮M, 15 ␮M, and 20 ␮M curcumin, the IC50 of imatinib (Supplementary Fig. 1). These results suggested that the cytotoxic-
decreased to 0.64 ± 0.09 ␮M, 0.40 ± 0.09 ␮M, 0.19 ± 0.06 ␮M, and ity curcumin was cancer selective, but not cell line specific.
0.06 ± 0.02 ␮M, respectively (Fig. 1D), and the cell viability declined Next, we examined the effects of curcumin to apoptosis in SUP-
to 42.61 ± 3.71%, 35.22 ± 3.54%, 26.56 ± 6.64%, and 16.52 ± 5.41%, B15 cells. The changes in cellular morphology were observed using
respectively (Fig. 1E). The interaction index (I) for 50% inhibition a light microscope. As shown in Fig. 1F, the membranes of the
of cell growth was 0.68, 0.65, 0.64, and 0.70 for 5 ␮M, 10 ␮M, untreated cells were lubricious and intact, while the membranes
15 ␮M, and 20 ␮M curcumin, respectively. These results indicated of cells treated with 30 ␮M curcumin were ruffled and lobulated in
there was a synergistic inhibitory effect of curcumin and imatinib. appearance. In comparison with 15 ␮M curcumin or 1 ␮M imati-
According to the above results of MTT cytotoxic assay, in our next nib treatment alone, the cells treated with curcumin plus imatinib
studies, we chose 30 ␮M and 1 ␮M as the major working concentra- showed more significant morphological changes. To further con-
tion of curcumin and imatinib, which were close to their IC50 values. firm apoptosis in SUP-B15 cells, the percentage of apoptotic cells

Fig. 3. Curcumin regulates the constitutive activation of the signaling pathways in SUP-B15 cell line. Representative western blots and densitometric analyses (expressed
as relative value of control) of SUP-B15 whole cell lysates are shown. The AKT/mTOR and RAF/MEK/ERK pathways, and the ABL, STAT5 and LYN protein kinases were
constitutively active in SUP-B15 cells, compared to normal human peripheral blood mononuclear cells (A). The cells were incubated with 30 ␮M curcumin for 3, 6, 12, and
24 h or with 10, 20, and 40 ␮M curcumin for 24 h. Cells were then harvested and total proteins were extracted. Equal amounts of proteins were separated on a 6-15% SDS-PAGE
gel and immunoblotted with the indicated antibodies. GAPDH was used as a loading control. Curcumin inhibited the constitutive activation of the AKT/mTOR (B and C) and
ABL/STAT5 (E and F) signaling pathways in a time- and dose-dependent manner. Curcumin had no inhibitory effects on the activation of PTEN, PDK1 (B and C) and LYN kinase
(E and F). RAF/MEK/ERK signaling was promoted by curcumin in early stage of exposure (at 3 h or 6 h), then declined gradually to base line (D). The results shown were
representative of two independent experiments. For the blots that the differences between the bands are not easy to be observed by bare eyes, the numbers labeled below
each blot represent relative intensity of the bands (B–F). The intensity values of the loading controls are not labeled.
6 Y. Guo et al. / The International Journal of Biochemistry & Cell Biology 65 (2015) 1–11

Fig. 4. The effect of Curcumin on the ratio of the expression of BCL2 to BAX and the effects of imatinib on the signaling pathways in SUP-B15 cell line. SUP-B15 cells were
incubated with 30 ␮M curcumin for 3, 6, 12, and 24 h or with 10, 20 and, 40 ␮M curcumin for 24 h. The cells were then harvested and total proteins were extracted to examine
the expression of BAX and BCL2 by Western Blots, and Ratio of the expression of BCL2 to BAX obtained from the mean densities (A and B). SUP-B15 cells were incubated
with 1 ␮M curcumin for 6, 12, and 24 h, then were harvested and total proteins were extracted to examine the expression of signaling protein, and densitometric analyses
of protein expression were performed. Curcumin up-regulated BAX expression significantly and down-regulated BCL2 mildly, which resulted in a reduced BCL2/BAX ratio in
the SUP-B15 cells (A and B). Imatinib inhibited the activation of MEK/ERK, ABL/STAT5 signaling (D and E), had no significant effect on the activation of LYN kinase (D), and
promoted the activation of AKT/mTOR signaling (A and E).The results shown were representative of two independent experiments. The numbers labeled below each blot
represent relative intensity of the bands (C–E). The intensity values of the loading controls are not labeled.

was analyzed by flow cytometry. The results indicated that apop- curcumin for 3 h, 6 h, 12 h, and 24 h, or to 10 ␮M, 20 ␮M and 40 ␮M
totic percent was 5.12 ± 0.74% in the control group and that it of curcumin for 24 h, after which the expression of the signaling
increased to 22.30 ± 4.41% and 53.23 ± 4.07% following treatment proteins was examined using Western Blotting. Our results indi-
with 15 ␮M and 30 ␮M curcumin for 24 h, respectively. Imatinib cated that curcumin down-regulated the constitutive activation of
alone educed cytotoxic effects dilatorily and had no significant ABL, AKT/mTOR (Fig. 3B and C) and STAT5 (Fig. 3E and F) in time- and
effects on apoptosis percent at 24 h. However, treatment with dose-dependent manners. However, curcumin had few inhibitory
1 ␮M imatinib and 15 ␮M curcumin resulted in an increase in the effects on PDK1 and PTEN (Fig. 3B and C) and no effects on LYN
percentage of apoptotic cells from 10.27 ± 1.77% to 41.53 ± 1.58% tyrosine kinase (Fig. 3E and F). As shown in Fig. 3D, RAF/MEK/ERK
(Fig. 2A). These results suggest that curcumin exerts a synergis- signaling was promoted by curcumin in early stage of exposure
tic effect with imatinib in inducing apoptosis in SUP-B15 cells. As (at 3 h, 6 h), then declined gradually to base line. In non-BCR/ABL
shown in Fig. 2B, curcumin also induced apoptosis in CEM and positive leukemia cell line CEM, curcumin inhibited the AKT/mTOR
THP-1 cells, the percentage of apoptotic cells was 38.92 ± 3.76%, and RAF/ERK signaling pathways (Supplementary Fig. 2), and in
40.13 ± 4.01%, respectively. Curcumin induced few apoptosis in THP-1 cells, curcumin inhibited the AKT/mTOR and RAF/MEK/ERK
normal human PBMCs, the percentage of apoptotic cells was all signaling activation simultaneously (Guertin and Sabatini, 2007).
lower than 10%. As PBMCs from healthy donors were negative for ABL/AKT/mTOR
and RAF/MEK/ERK signaling activation (Fig. 3A), so we did not
examined the effects on them. Above results indicated that anti-
3.2. Curcumin down-regulates the constitutive activation of ABL leukemia effects of curcumin were not BCR/ABL kinase dependent.
kinase, AKT/mTOR pathway and STAT5 kinase in SUP-B15 cells

BCR/ABL, SRC kinases and their downstream signaling path- 3.3. Curcumin induces a reduced BCL2/BAX ratio in the SUP-B15
ways including AKT/mTOR, RAF/MEK/ERK and STAT5, are involved cells
in controlling the proliferation, differentiation and apoptosis of
Ph+ ALL cells. To study the mechanisms of anti-leukemia effects The deregulated expression of apoptosis mediators, such as
of curcumin, we first examined their activation levels in SUP-B15 BCL2 and BAX, may disrupt apoptosis signaling in leukemia cells.
cells. As shown in Fig. 3A, the ABL kinase, LYN kinase, AKT/mTOR, To explore the effects of curcumin on BCL2 and BAX, SUP-B15 cells
RAF/MEK/ERK, and STAT5 signaling pathways are constitutively were exposed to the indicated concentration of curcumin for differ-
active in SUP-B15 cells. To investigate the effects of curcumin on ent time periods, and the expression of BCL2 and BAX was examined
these signaling pathways, SUP-B15 cells were exposed to 30 ␮M of with a Western Blot assay. The results showed that curcumin
Y. Guo et al. / The International Journal of Biochemistry & Cell Biology 65 (2015) 1–11 7

Fig. 5. Curcumin inhibited the up-regulation of AKT/mTOR pathway mediated by imatinib in SUP-B15 cells. SUP-B15 cells were treated with 1 ␮M imatinib alone, or in
combination with 15 ␮M curcumin for 6 h, 12 h, and 24 h. Then, total proteins were extracted and the effects on the growth signaling pathways were examined by Western
Blot with indicated antibodies, and densitometric analyses (expressed as relative value of control) of protein expression were performed. The imatinib-mediated up-regulation
of AKT/mTOR signaling was inhibited by the addition of 15 ␮M curcumin. Curcumin couldn’t inhibit the imatinib-mediated activation of RAF, and had no synergistic inhibitory
effect on MEK signaling. The numbers labeled below each blot represent relative intensity of the bands. The intensity values of the loading controls are not labeled.

augmented BAX significantly and reduced BCL2 expression mildly (Fig. 4D and E). However, imatinib up-regulated the AKT/mTOR
in a time- and dose-dependent manner, which resulted in a reduced signaling pathway (Fig. 4C). Meanwhile, our results indicated that
BCL2/BAX ratio (Fig. 4A and B), as it is an important indicator of curcumin down-regulated the activation of AKT/mTOR in SUP-B15
apoptosis. cells. Thus, we investigated whether curcumin could overcome
the activation of AKT/mTOR signaling mediated by imatinib. SUP-
3.4. Curcumin inhibits the up-regulation of AKT/mTOR pathway B15 cells were exposed to 1 ␮M imatinib alone or in combination
mediated by imatinib in SUP-B15 cells with 15 ␮M curcumin for 6 h, 12 h and 24 h, and the expression
of signaling proteins were examined. Fig. 5 shows that combining
Our data showed that imatinib exerted its anti-leukemia effects curcumin and imatinib inhibited the up-regulation of AKT/mTOR
by inhibiting MEK/ERK and ABL/STAT5 signaling in SUP-B15 cells signaling that was mediated by imatinib. However curcumin could

Fig. 6. Curcumin down-regulated the BCR/ABL gene expression in SUP-B15 cells. The cells were treated with 1 ␮M imatinib, 20 ␮M curcumin, 30 ␮M curcumin, 40 ␮M
curcumin, or 1 ␮M imatinib plus 15 ␮M curcumin for 24 h, and total RNA was isolated to measure BCR/ABL gene expression by semi-quantitative RT-PCR. ␤-Actin was used
as an internal control. Curcumin down-regulated the BCR/ABL gene expression in SUP-B15. The results shown were representative of two independent experiments.
8 Y. Guo et al. / The International Journal of Biochemistry & Cell Biology 65 (2015) 1–11

Fig. 7. Curcumin down-regulates the signaling pathways in Ph+ ALL patient primary cells. The mononuclear cells from five Ph+ ALL patients were treated with the indicated
concentration of curcumin and imatinib alone or in combination for 24 h. The activation status (phosphorylation) of signaling proteins was detected with a Western Blot.
Curcumin significantly suppressed the AKT/mTOR, RAF/MEK, STAT5 and LYN signaling pathways in all five primary specimens. In patient 4, who progressed under imatinib
therapy, treatment with 10 ␮M imatinib resulted in a slight inhibition of the AKT/mTOR, RAF/MEK, STAT5 and LYN signaling pathways. However, 10 ␮M imatinib plus 20 ␮M
curcumin significantly inhibited the activation of the mTOR and STAT5 signaling pathways, while the synergetic inhibitory effects on RAF/MEK and LYN signaling pathways
were weak.

not inhibit the imatinib-mediated activation of RAF, and had no Our results indicated that curcumin had cytotoxic effects on all
synergistic inhibitory effect on MEK signaling. five patient specimens. The IC50 at 72 h was 29.45 ␮M, 20.03 ␮M,
35.33 ␮M, 36.22 ␮M, and 24.45 ␮M, and the cell viability after
3.5. The effects of curcumin on the mRNA levels of the BCR/ABL 40 ␮M curcumin treatment for 72 h was 17.91%, 14.80%, 35.15%,
gene in SUP-B15 cells 42.14% and 19.51%, respectively. In the primary cells from patient
4, who was resistant to imatinib, the IC50 of imatinib alone was
The BCR/ABL fusion gene generated by reciprocal translocation 15.85 ␮M, and decreased to 5.79 ␮M when treated in combination
is the root cause of Ph+ leukemia. Thus, to further investigate the with 20 ␮M curcumin. The interaction index (I) for 50% inhibition
anti-leukemia mechanisms of curcumin, we explored its effects on of cell growth was 0.92, which indicated a synergetic anti-leukemia
BCR/ABL gene expression in SUP-B15 cells using RT-PCR. As shown effect of curcumin and imatinib in the specimen from the patient
in Fig. 6, curcumin suppressed the BCR/ABL gene expression, while who was resistant to imatinib.
imatinib alone had no significant effects on the mRNA levels of Because of limitations in the number of samples obtained from
the BCR/ABL gene. However, imatinib plus curcumin significantly the patients, these cells were only incubated with 40 ␮M cur-
down-regulated the BCR/ABL gene expression. cumin for 24 h to measure the signaling protein changes, and the
synergetic effect of curcumin and imatinib was only examined in
3.6. Curcumin induces cytotoxicity effects in Ph+ ALL primary the imatinib resistant patient. As shown in Fig. 7, the AKT/mTOR,
cells by suppressing signaling pathways RAF/MEK, and STAT5 signaling pathways were all constitutively
active in all five patient specimens. These results were similar to
The characteristics of Ph+ ALL patients are not fully represented what we observed in the SUP-B15 cells. However, in contrast to
by cell lines. Therefore, we investigated the anti-leukemia effects the SUP-B15 cells, in the patient specimens, curcumin not only
and mechanisms of curcumin action on primary human Ph+ ALL inhibited the AKT/mTOR and STAT5 signaling but also markedly
cells. Bone marrow samples from five Ph+ ALL patients were stud- inhibited the RAF/MEK signaling. In patient 4, who progressed
ied. Of the five patients, patient 1, patient 2, and patient 5 were under imatinib therapy, we only observed slight inhibitory effects
newly diagnosed; patient 3 relapsed after the discontinuation of on these three signaling pathways, even when the concentration
imatinib therapy, and patient 4 progressed under imatinib treat- of imatinib used was as high as 10 ␮M. However, 40 ␮M curcumin
ment. was able to significantly suppress all three signaling pathways. In
Y. Guo et al. / The International Journal of Biochemistry & Cell Biology 65 (2015) 1–11 9

Fig. 8. The in vivo anti-leukemia role of curcumin in Ph+ ALL xenograft mice. Semi-quantitative RT-PCR analysis showed a down-regulation of BCR/ABL mRNA in the bone
marrow of mice treated with 50 mg/kg day curcumin, while the down-regulation of BCR/ABL mRNA in 5 mg/kg day imatinib treatment group was slight. However, a significant
reduction in BCR/ABL mRNA was found in mice treated with the combination of curcumin at 25 mg/kg day and imatinib at 5 mg/kg day (A). The expression of CD19 in the
spleen was assayed using immunohistochemistry. Curcumin significantly decreased the leukemic infiltration in the spleen. Additionally, the combination of imatinib and
curcumin had a synergistic role in the decrease of leukemic infiltration in the spleen (B).

contrast to imatinib alone, the combination of 10 ␮M imatinib and expression of BCL2 to BAX, and down-regulating BCR/ABL gene
20 ␮M curcumin inhibited these signaling pathways. expressions. Furthermore, we found that in the SUP-B15 cell line,
curcumin exerted a synergetic anti-leukemia effect with imatinib
3.7. Curcumin inhibits growth of leukemia cells in Ph+ ALL by inhibiting the expression of BCR/ABL gene and the hyperacti-
xenograft mice vation of AKT/mTOR signaling mediated by imatinib. In primary
samples from Ph+ ALL patients, curcumin inhibited growth not
Because all studies on the anti-leukemia effects of curcumin on only in the cells from newly diagnosed patients but also in cells
Ph+ ALL cells have been performed in vitro, we investigated the from an imatinib-resistant patient. Moreover, curcumin exhibited
in vivo efficacy of curcumin using our mouse model of Ph+ ALL. anti-leukemia effects and showed a synergetic anti-leukemia effect
As shown in Fig. 8, in mice treated with 50 mg/kg day curcumin, with imatinib in a xenograft leukemia model established with SUP-
the mRNA levels of BCR/ABL gene in the bone marrow decreased B15 cells. These results demonstrate that curcumin might be a
significantly. This was accompanied by a decrease in leukemic infil- promising agent for the treatment of patients with Ph+ ALL and that
tration in the spleen, which indicated the reduction of the leukemia curcumin might be particularly effective when used with current
burden in vivo. Compared to treatment with 5 mg/kg day imatinib induction regimens including imatinib to treat Ph+ ALL.
alone, the reduction of leukemic infiltration in the bone marrow and The constitutive active BCR/ABL kinase is the trigger of Ph+
the spleen was more significant in mice treated with combination leukemia. The BCR/ABL inhibitor, imatinib, has markedly altered
of 25 mg/kg day curcumin and 5 mg/kg day imatinib. These results the management of Ph+ leukemia. However, imatinib efficacy is
demonstrated that curcumin had significant in vivo anti-leukemia much lower in Ph+ ALL, compared with CML, and imatinib resis-
efficacy, a result that correlated with our in vitro results; curcumin tance and relapse are continuous problems. The mechanism for
exerted synergistic anti-leukemia effects with imatinib in vivo. resistance to imatinib has mainly been ascribed to mutations in the
BCR/ABL kinase domain; whereas the independent BCR/ABL mech-
4. Discussion anisms are contribute to resistance less frequently. Our previous
studies have shown that in SUP-B15 cells, the level of constitu-
Our present study shows that curcumin induced cytotoxic tive activation of the AKT/mTOR pathway was much higher than in
effects on a Ph+ ALL cell line by down-regulating the activation K562 cells (Guo et al., 2012). Our present results showed that treat-
of AKT/mTOR, and ABL/STAT5 pathways, reducing the ratio of the ment with imatinib up-regulated the activation of the AKT/mTOR
10 Y. Guo et al. / The International Journal of Biochemistry & Cell Biology 65 (2015) 1–11

pathway in SUP-B15 cells and induced little apoptosis at 24 h. These to draft the manuscript. All authors read and approved the final
findings led us to speculate that the activation of the AKT/mTOR manuscript.
signaling was independent of the BCR/ABL tyrosine kinase, and this
pathway was more crucial than others in sustaining cell survival in
Acknowledgements
Ph+ ALL. We also speculated that the hyper-activation of AKT/mTOR
signaling may be one reason of lower imatinib response and one
This work was supported by the grants from National Natural
cause of imatinib resistance in Ph+ ALL.
Science Foundation of China (no. 30770912 and no. 81400123),
Considering the master regulatory function of AKT/mTOR
Foundation of the Science & Technology Department of Sichuan
signaling in cancer cells (Guertin and Sabatini, 2007) and its impor-
Province (no. 2013SZ0025) and Doctoral Tutor Fund of Ministry of
tant role in the survival in SUP-B15 cells, we speculated that the
Education (no. 20120181110008).
AKT/mTOR signaling inhibitors would significantly suppress the
proliferation of SUP-B15 cells and induce apoptosis. Moreover, they
may potentiate the efficacy of imatinib in Ph+ ALL. Many reports Appendix A. Supplementary data
have indicated that curcumin exhibits cytotoxicity in many cancer
cell lines by targeting the AKT/mTOR pathway (Woo et al., 2003; Supplementary data associated with this article can be found,
Bava et al., 2005; Aggarwal et al., 2006; Beevers et al., 2006). There- in the online version, at http://dx.doi.org/10.1016/j.biocel.2015.05.
fore, we hypothesized that curcumin alone can exert anti-leukemia 003
effects in Ph+ ALL by inhibiting AKT/mTOR signaling. Addition-
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