Guaianolide sesquiterpene lactones, a source to discover agents that selectively inhibit acute myelogenous leukemia stem and progenitor cells

Article abstract of DOI:10.1021/jm301064b

Small molecules that can selectively target cancer stem cells (CSCs) remain rare currently and exhibit no common structural features. Here we report a series of guaianolide sesquiterpene lactones (GSLs) and their derivatives that can selectively eradicate

Full text of DOI:10.1021/jm301064b

Article
pubs.acs.org/jmc
Guaianolide Sesquiterpene Lactones, a Source To Discover Agents
That Selectively Inhibit Acute Myelogenous Leukemia Stem and
Progenitor Cells
Quan Zhang,†,# Yaxin Lu,†,# Yahui Ding,†,# Jiadai Zhai,†,# Qing Ji,†,# Weiwei Ma,† Ming Yang,†
Hongxia Fan,† Jing Long,† Zhongsheng Tong,§ Yehui Shi,§ Yongsheng Jia,§ Bin Han,∥
Wenpeng Zhang,∥ Chuanjiang Qiu,∥ Xiaoyan Ma,∥ Qiuying Li,∥ Qianqian Shi,⊥ Haoliang Zhang,†
Dongmei Li,† Jing Zhang,† Jianping Lin,† Lu-Yuan Li,† Yingdai Gao,*,† and Yue Chen*,†
†College of Pharmacy and The State Key Laboratory of Elemento-Organic Chemistry, Nankai University, and State Key Laboratory of
Experimental Hematology, Institute of Hematology, Chinese Academy of Medical Sciences, Tianjin, People's Republic of China
§Medical Department of Breast Oncology, Tianjin Medical University Cancer Institute and Hospital, Tianjin 300060,
People's Republic of China
∥Accendatech Company, Ltd., Tianjin 300384, People's Republic of China
⊥College of Food Engineering and Biotechnology, Tianjin University of Science and Technology, Tianjin 300457,
People's Republic of China
S
* Supporting Information
ABSTRACT: Small molecules that can selectively target cancer
stem cells (CSCs) remain rare currently and exhibit no common
structural features. Here we report a series of guaianolide ses-
quiterpene lactones (GSLs) and their derivatives that can
selectively eradicate acute myelogenous leukemia (AML) stem
or progenitor cells. Natural GSL compounds arglabin, an anti-
cancer clinical drug, and micheliolide (MCL), are able to reduce
the proportion of AML stem cells (CD34+CD38−) in primary
AML cells. Targeting of AML stem cells is further confirmed by
a sharp reduction of colony-forming units of primary AML cells
upon MCL treatment. Moreover, DMAMCL, the dimethylami-
no Michael adduct of MCL, slowly releases MCL in plasma and
in vivo and demonstrates remarkable therapeutic efficacy in the
nonobese diabetic/severe combined immunodeficiency AML models. These findings indicate that GSL is an ample source for
chemical agents against AML stem or progenitor cells and that GSL is potentially highly useful to explore anti-CSC approaches.
and parthenolide (PTL)9 (Figure 1), but none of them are
currently on the market as anticancer drugs. Chang reported
INTRODUCTION
■
Cancer stem cells (CSCs) are able to differentiate into cancer
that lapatinib treatment prevented the increase of tumorigenic
progenitor cells and further into other cell types in a cancer
cells in patients with locally advanced breast cancer and that a
context. It was first discovered that CD34+CD38− leukemia stem
remarkable response rate was observed in a combination therapy
cells could form tumors when transplanted into the SCID mouse
approach using lapatinib, trastuzumab, and decetaxel.10
model.1 Many types of CSCs in solid tumors have been
Novel anti-CSC compounds are highly desirable not only
for therapeutic purposes, but also for mechanistic studies to
identified, including cancers of the brain, colon, ovary, pancreas,
and prostate and melanoma.2 It is now widely accepted that
understand stem cell biology. Although a number of intracellular
CSCs play a key role in carcinogenesis, cancer disease pro-
gression, relapse, and drug resistance.3 Conventional chemo-
signals and transcription factors such as Bmi-1, Notch, Sonic
hedgehog, Wnt, NF-κB, and telomerase have been proposed to
therapies often lead to increased proportions of CSCs in
cancers.4a CSC-enriched cell populations not only demonstrate
be potential targets for the modulation of CSC activities, these
signaling pathways are shared with normal stem cells.11 New
escalated resistance to chemotherapeutic agents and ionizing
radiation,4 but also exhibit elevated tumor-forming potential.4a
entities that selectively regulate CSC activities are urgently
needed as probes for signaling pathways specific to CSCs.
However, chemical compounds that selectively inhibit CSC growth
are rarely reported. Thus far, only a small number of compounds
have been reported to selectively inhibit CSCs, including
Received: July 20, 2012
salinomycin,4 CITCO,5 repertaxin,6 MG-132,7 sulforaphane,8
© XXXX American Chemical Society
A
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Figure 1. Structures of anti-CSC small molecules.
Parthenolide (PTL), a natural sesquiterpene lactone (SL) of
10,5-ring structure isolated from Tanacetum parthenium
(Feverfew), is among the known anti-CSC molecules extensively
studied.9,11−13 SLs are a family of chemical structures with more
than 5000 members, many of which are used in traditional
Chinese medicine for treatment of inflammation.14 Some studies
indicate that the anti-inflammatory activities of SL are related to
the inhibition of the transcription factor NF-κB.15 PTL has been
found to target NF-κB, Stat3, HDAC, SERCA, and COX-2 in
cancer cells.12 The inhibition of NF-κB and introduction of
reactive oxidative species (ROS) appear to be important for PTL
activity against CD34+CD38− leukemia stem cells (LSCs)9a,b
and breast cancer stem cells (BCSCs).9d The water-soluble
Michael adduct of PTL, DMAPT,9f has entered clinical trial for
cancer therapy.9g However, PTL is unstable in both acidic and
basic conditions.16
We have studied the activities of a series of SLs against AML
stem or progenitor cells. Our data demonstrate that the 10,5-ring
structure of PTL is not essential to the AML growth inhibition
activity and that certain guaianolide sesquiterpene lactones
(GSLs), a subtype of SLs characterized by a 5,7,5-ring
structure,17 can selectively inhibit AML stem or progenitor cell
growth. Moreover, a Michael adduct of micheliolide (MCL),
dimethylaminomicheliolide (DMAMCL), exhibits remarkable
therapeutic efficacy in AML NOD/SCID murine models.
to its interaction with biological nucleophiles, such as cysteine
sulfhydryl groups of target proteins, by a Michael-type
addition,20−22 we also analyzed α-methylene-γ-lactone (4),
itaconic anhydride (5), andrographolide (6), sarmentine (7),
and a series of synthesized SL analogues (8−22) for their
anticancer activities.
Activities against Cultured AML Cell Lines. The
compounds were assayed against cultured AML cell line HL-60
and doxorubicin-resistant cell line HL-60/A (Table 1).
Doxorubicin (DOX) and PTL were used as positive controls.
The N3 Michael adduct of PTL (14) demonstrated an activity
profile similar to that of PTL, whereas the triazole derivatives
of 14 (compounds 15−18) were inactive. Further analysis
indicated that the N3 adducts were completely converted to PTL
in 3 h at physiological pH (7.4), whereas the inactive compounds
15−18 did not release PTL in 24 h under these experimental
conditions. Additionally, the other 10,5-ring compound
costunolide 2 was slightly less active than PTL; this suggests
that the epoxide moiety of PTL is not essential for these
biological activities.
Compounds 1, 8−10, 12−13, and 19−22 are a group of
structures with a 5,7,5-ring. Among these compounds, 1, 8−10,
12, and 19−21 exhibited activities against HL-60 and HL-60/A
comparable to that of PTL. Compound 13 (C10-OH-
substituted) was less potent in comparison. However, the
product with α-methylene reduction (compound 22) was
completely inactive, indicating that α-methylene-γ-lactone is
necessary for the anti-AML activity. Other non-SL natural
products (compounds 4−7) and an open-ring SL derivative
(compound 11) with Michael acceptors were basically inactive
against HL-60 and HL-60/A cell lines. These results indicate
that the 10,5- or 5,7,5-ring is important for SLs’ anti-AML cell
activities. For all the active SLs and derivatives, the activity
against drug-resistant cell line HL-60/A was comparable to that
against sensitive cell line HL-60. Compared with DOX, which
exhibits an IC50 of more than 100-fold against HL-60/A and
RESULTS
■
Compound Syntheses. The compounds contain α,β-
unsaturated carbonyl structures, including SLs and their
derivatives, as shown in Figures 2−4. In comparison with PTL,
several other commercially available SLs, including arglabin,18
dehydrocostus lactone (1), and costunolide (2), as well as
artemsinin (3), which was reported to generate ROS to eradicate
cancer cells,19 were evaluated for their anticancer activities.
Since it is generally perceived that the α-methylene-γ-lactone
moiety of SLs is responsible for their biological activities due
B
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Figure 2. Structures of arglabin and compounds 1−13.
Figure 3. Synthesis of compounds 14−18.
progenitor cells vs total leukemia cells after treatment with these
compounds were (arglabin) 10.7% vs 20.0%, (1) 3.8% vs 6.4%,
(2) 7.5% vs 13.4%, (9) 2.3% vs 8.4%, (10) 44.5% vs 49.7%, (12)
22.6% vs 30.6%, (14) 17.5% vs 22.8%, (19) 2.1% vs 10.8%, and
(20) 11.2% vs 53.1%. These data indicate that the survival rates
of AML stem/progenitor cells were lower than those of total
leukemia cells upon treatment with these nine compounds. Again
in comparison, treatment with DOX resulted in significantly
higher survival rates for AML stem/progenitor cells vs total
leukemia cells (115.0% vs 37.6% at 5 μM and 49.1% vs 32.5% at
10 μM), commonly observed also for other traditional anticancer
agents.1−3
Compounds arglabin, 1, 8−10, 10−13, and 19−22 are GSLs
with characteristic 5,7,5-ring moieties. Since compound 9
(MCL) showed high potency and selectivity against leukemia
stem/progenitor cells, it was used as a scaffold for a preliminary
structure−activity relationship (SAR) study (Figure 5). The
A-region-modified compound 22, derived from reduction of
the 11,13-double bond of MCL, exhibited diminished inhibi-
tory activity on both total leukemia cells and leukemia stem/
progenitor cells (Table 2). This indicates that the α-methylene-
γ-lactone is essential to the anti-AML activity, which is consistent
with that of parthenolide.9f The B-region-modified compounds
8, 10, 12, and 13 maintained activities against HL-60 and
HL-60/A comparable to those of MCL (Table 1); however,
their inhibitory activities against AML stem/progenitor cells were
reduced. For example, compound 8 (C10-OCH3-substituted),
Figure 4. Synthesis of compounds 19−22.
HL-60, these active SLs are considerably more potent against
drug-resistant AML cells.
Activities against Primary AML Cells and Stem/
Progenitor Cells. Compounds 1, 2, 8−14, and 19−22 and
arglabin were screened in the assay against primary total
leukemia cells and leukemia stem/progenitor cells (CD34+)
isolated from blood samples of AML patients (Table 2). The cell
viability was determined by annexin labeling after 18 h of
treatment. At 10 μM, compounds arglabin, 1, 2, 9, 10, 12, 14, and
19−20 showed significant activity against leukemia stem/
progenitor cells (CD34+-labeled). The cell viabilities of stem/
C
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a
Table 1. Biological Activities of Compounds against Cultured AML Cells (HL-60 and HL-60/A)
compd
HL-60 IC50, μM
HL-60/A IC50, μM
compd
HL-60 IC50, μM
HL-60/A IC50, μM
b
DOX
0.05 0.01
3.7 0.1
5.3 1.7
7.7 1.2
>50
6.7 0.8
4.1 0.4
3.7 0.9
5.8 1.4
>50
11
12
13
14
15
16
17
18
19
20
>50
>50
PTL
9.4 2.6
21.9 3.2
4.3 0.7
>50
9.9 2.1
17.9 1.2
5.9 0.55
>100
1
2
3
4
35.0 3.9
>50
28.8 5.4
>50
>50
>100
5
>50
>100
6
27.0 1.5
>50
23.3 6.4
>50
>50
>100
7
9.9 0.9
6.2 1.7
17.2 1.1
>50
10.2 0.14
6.7 2.1
21.5 2.0
>50
8
9.4 0.8
5.5 1.4
4.5 0.8
11.2 1.6
6.2 2.2
9 (MCL)
21(DMAMCL)
10
6.4 0.3
22
a
b
All values are the mean of three independent experiments. DOX = doxorubicin, an anticancer agent used as a positive control.
Table 2. Biological Activities of Compounds against Total Cancer Cells and Cancerous Stem/Progenitor Cells (CD34+-Labeled)
from AML Patient Blood Samples
5 μM
10 μM
total
CD34+
total
CD34+
a
a
a
a
compd
DOX
av % viable
SD
av % viable
SD
av % viable
SD
av % viable
SD
b
b
c
c
37.6
4.2
1.1
115.0
13.2
3.4
0.9
32.5
2.3
0.4
1.5
0.2
0.5
0.3
0.5
2.6
0.7
1.3
2.9
3.2
0.5
1.0
0.1
1.2
49.1
4.7
4.5
0.4
0.9
0.3
0.1
0.5
0.6
2.4
0.4
1.8
2.7
5.7
0.2
1.0
0.1
1.0
PTL
18.0
8.3
20.0
6.4
arglabin
10.7
3.8
1
6.1
25.8
96.5
11.7
70.7
83.2
61.6
88.5
31.5
34.1
64.3
77.6
85.0
0.3
1.1
0.7
2.2
0.5
1.4
1.0
6.6
0.9
0.9
9.9
3.0
1.4
2.9
18.8
95.8
4.2
0.2
1.0
0.7
2.2
1.1
1.1
2.5
8.9
1.1
1.5
3.3
2.7
2
13.4
75.2
8.4
7.5
8
76.3
2.3
9 (MCL)
10
67.9
81.5
53.7
84.9
28.4
25.5
26.3
72.9
83.1
49.7
72.0
30.6
89.9
22.8
10.8
53.1
64.9
70.8
44.5
70.9
22.6
84.8
17.5
2.1
11
12
13
14
19
20
11.2
60.4
74.4
21(DMAMCL)
22
1.5
a
b
c
Viability normalized to untreated controls. DOX was treated with 0.1 μM. DOX was treated with 0.5 μM.
largely maintained activity against AML stem/progenitor cells
(2.1% viable CD34+ cells at 10 μM) comparable to that of MCL
(2.3% viable CD34+ cells at the same concentration). Arglabin,
the compound with the hydroxyl group eliminated, also showed
high potency against leukemia stem/progenitor cells (10.7%
viability at 10 μM). These findings suggest that the hydroxyl
group can accommodate certain types of modifications, such
as etherification or elimination. Compound 1, dehydrocostus
lactone, which has an olefin in the C-region and a methylene
moiety in the B-region, demonstrated activities against both total
leukemia cells and leukemia stem/progenitor cells comparable
to those of MCL, but was unstable at room temperature. We
therefore selected MCL and arglabin for further evaluation.
MCL and Arglabin Selectively Inhibit Leukemia Stem
Cells. We analyzed the effects of PTL, MCL, and arglabin treat-
ment on CD34+CD38− AML cells. The percentages of CD34+
and CD34+CD38− cells in all surviving cells in response to these
compounds at 10 μM are given in Table 3. The proportions of
stem cells in all living cells decreased by 2.6-, 1.9-, and 5.3-fold for
PTL, arglabin, and MCL treatment, respectively, compared with
those in the vehicle-treated controls. This finding indicates that
Figure 5. Preliminary structure−activity relationship of 5,7,5-ring-type
SLs.
a GSL modified at the B-region with high activity against
HL-60 (IC50 = 9.4
0.8 μM) and HL-60/A (11.2
1.6 μM) cell lines, showed weak potency against human AML
stem and progenitor cells (76.3% viable CD34+ cells at a
concentration of 10 μM). This suggests that certain types of
modifications in the B-region affect the anti-AML cell activity,
especially the AML stem and progenitor cells. For the C-region,
methylation of the hydroxyl group of MCL (compound 19)
D
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Table 3. Proportion of CD34+ and CD34+CD38− Cells in
Overall Living Cells in Response to Compounds PTL,
Arglabin, and MCL at a Concentration of 10 μM
MCL Selectively Inhibits AML-Subtype Cell Lines
Obtained from Human Patients. We further analyzed PTL
and MCL against primary AML cells isolated from blood samples
of AML patients (Figure 6) using methods as described.9a At a
concentration of 10 μM MCL, lower cell viability was observed
for the CD34+ subtype than that for total AML cells. This indi-
cates that MCL, similar to PTL, may selectively inhibit AML
progenitor/stem cells.
Effect of MCL on Colony-Forming of AML Cells. The
mononuclear cells from cord blood of AML patients were cul-
tured in serum-free Iscove's modified Dulbecco's medium
(IMDM) for 18 h in the presence of DOX, PTL, and MCL,
respectively. Since DOX has a much lower IC50 concentration
against AML cells than PTL and MCL (Table 1), we used low
concentrations of DOX (0.05 and 0.1 μM) to ensure a com-
parable proportion of AML cells survived after drug treatments.
fold
decrease
CD34+CD38−
(%)
fold
decrease
a
b
compd
CD34+ (%)
control
PTL
57.9 2.6
15.1 4.2
31.7 0.7
17.8 2.5
1
12.1 3.3
4.6 0.8
6.4 0.1
2.3 0.9
1
3.8
1.8
3.2
2.6
1.9
5.3
arglabin
MCL
a
Ratio of the percentage of CD34+ cells compared to that of the
b
control. Ratio of the percentage of CD34+CD38− cells compared to
that of the control.
the three compounds selectively eradicate AML progenitor/stem
cells and stem cells.
Figure 6. Analysis of PTL- and MCL-induced apoptosis with flow cytometry. (a) Viability of CD34+ cells in a typical leukemia specimen in response to
compounds PTL and MCL. Q1 indicates the necrotic cells, Q2 indicates the late apoptosis of the leukemia cells, Q3 indicates the early apoptosis of the
leukemia cells, and Q4 indicates the viability of leukemia cells. From the pictures we can conclude that the cells of Q4 were reduced strongly after the
treatment of PTL and MCL, which indicated the viabilities of the cells were strongly reduced. (b) Viability (Q1) of total cells, CD34+-labeled cells, and
CD34+CD38−-labeled cells in various leukemia specimens in response to compounds PTL and MCL at 10 μM.
E
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The numbers of colonies were scored after 15−20 days of
culture. The colony-forming units (CFUs) of DOX-treated
cells were 165−170 (Figure 7), approximately 25% reduction
NOD/SCID mice. The animals were sacrificed after 8 weeks,
and the percentage of human mononuclear cells (with a CD45+
maker) in the bone marrow and spleen was determined (Figure 9).
Detection of CD45+ cells indicates human leukemia cells engrafted,
and then it can be concluded that both PTL and MCL treatments
substantially reduced the ability of leukemia stem cells to engraft
in the animals in the bone marrow (p < 0.01, Figure 9a) and the
spleen (p < 0.01, Figure 9b). Human cancer cells were detected
in a significant number in the spleen of the control group, but
hardly were detectable in PTL- or MCL-treated groups. This
finding demonstrates that both PTL and MCL can reduce the
cancer-initiating capability of AML cells.
Stability in Mouse Plasma. The half-lives of PTL and MCL
in mouse plasma are given in Table 4. The half-life of PTL is only
0.34 h, whereas that of MCL is 2.64 h. These in vitro studies
suggest that the structure optimization from PTL to MCL has
improved the compound physiological stability by 7-fold; thus, it
is likely to be more stable in vivo.
Transformation from Prodrug to Active Drug. With the
similiar method to prepare DMAPT from PTL,9f we synthesized
the dimethylamino Michael adduct of MCL, DMAMCL, to
improve aquatic solubility (Figure 4, compound 21). DMAPT
and DMAMCL were analyzed in HEPES buffer as well as in
plasma. In HEPES with pH lower than 5.0, DMAPT and
DMAMCL were stable, and no significant amount of PTL or
MCL was released in 5 days (data not shown). However, in
HEPES with pH 7.4, DMAPT was converted to PTL rapidly,
while DMAMCL released MCL slowly but consistently (Figure 10).
In plasma, DMAMCL and DMAPT transformed to MCL and
PTL, respectively (Figure 11). The half-life of DMAMCL is
much longer; thus, MCL was continuously released from
DMAMCL steadily for more than 8 h. In contrast, the con-
centration of PTL released from DMAPT decreased to 0.1 μg/mL
in 2.5 h. These data indicate that DMAMCL has superior in vivo
kinetic properties compared to DMAPT as a prodrug, and this
advantage of DMAMCL might lead to better therapeutic
potential.
Pharmacokinetic Study of DMAMCL in Mice. The plasma
concentration−time curves for DMAMCL and MCL after a
single 150 mg/kg per os (po) dose of an aqueous solution of
DMAMCL in mouse are given in Figure 12a. The pharmaco-
kinetic parameters determined with a noncompartmental model
are given in Table 5. Having quickly reached a peak, the plasma
concentrations of both DMAMCL and MCL declined gradually.
Thus, DMAMCL was constantly transformed into MCL in vivo.
These findings indicate that DMAMCL can sustain the release of
Figure 7. Reduction of methylcellulose colony-forming in response to
compounds DOX, PTL, and MCL.
compared with that of the vehicle control. However, the CFUs
after PTL or MCL treatment were dramatically reduced on
average to 4.5 or lower. The significant reduction of AML CFUs
by PTL, but not that by MCL, was reported previously by other
investigators.9a These findings indicate that MCL, similar to
PTL, preferentially targets AML progenitor cells.
Effect of MCL on Normal Blood Cells and Hema-
topoietic Stem/Progenitor Cells. An in vitro apoptosis assay
was used to evaluate the effect of MCL on normal hematopoietic
stem/progenitor cells (HSCs). The percentage of apoptotic
cells was normalized to that of the vehicle control (Figure 8a).
MCL showed less effect against normal hematopoietic stem/
progenitor cells (CD34+ cells) than PTL (p < 0.05, Figure 8b). In
other words, MCL is potentially safer than PTL at the cell level.
Ex Vivo Biological Activity of MCL in an AML Murine
Model. We investigated the selectivity of MCL against AML
stem cells in an ex vivo experiment by using an NOD/SCID
murine model. Freshly isolated primary AML cells were treated
with saline, PTL, and MCL, respectively, for 18 h, and then the
same numbers of surviving cells were intravenously injected into
Figure 8. Normal specimens in response to compounds PTL and MCL. (a) Viability of total normal cells (mononuclear cells from the umbilical cord
blood samples) in response to compounds PTL and MCL at a concentration of 5 μM. (b) Viability of CD34+-labeled normal cells (stained by the
antibody CD34-allophycocyanin for 30 min) in response to compounds PTL and MCL at a concentration of 5 μM (the asterisk indicates p < 0.05).
F
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Figure 9. Analysis of CD45+CD33+ cells in bone marrow and spleen with flow cytometry. Percentage of CD45+ cells in the bone marrow (two asterisks
indicate p < 0.01) (a) and CD33+CD45+ cells in the spleen (two asterisks indicate p < 0.01) (b) in NOD/SCID mice, which were sacrificed 8 weeks after
◇
□
,
■
○
,
●
transplantation with the primary AML cells pretreated with PTL and MCL for 18 h. Each
,
, or symbol represents a single animal analyzed at
8 weeks after transplantation. The mean percentage is indicated by the horizontal bars. (c) Analysis of typical spleen samples with flow cytometry. The
CD33+CD45+ cells (in the circles) were obviously in the spleen in the control, but in PTL- and MCL-treated groups, the CD33+CD45+ cells were nearly
invisible in the spleen.
a
Table 4. Half-Life of PTL and MCL in Mouse Plasma
model. The model was established by tail vein injection of
primary AML cells isolated from AML M5 patients. After 8 weeks
postinoculation, 100 mg/kg DMAMCL or 100 mg/kg DMAPT
was orally administered once every other day for a total of
seven doses. As a control experiment, 1 mg/kg DOX was admi-
nistered via tail vein every other day for a total of four doses.
As shown in Figure 13b, the average life span of the DOX-
treated animals was 22 days, which is shorter than that of the
untreated control group (43 days). Similar results for DOX
treatment were observed in teh literature,23 and this is likely
due to sensitivity of the NOD/SCID mice to DOX toxicity.
The average life spans for DMAPT- and DMAMCL-treated
groups were 66 and 63 days, respectively. Moreover, 17% of
DMAPT-treated (1/6) and 33% of DMAMCL-treated (2/6)
animals remained alive on day 85 postinoculation. These three
animals were sacrificed, and the bone marrow was analyzed.
The percentage of CD45+CD33+ human AML cells in the
PTL
0.34
MCL
2.64
t1/2, h
a
The starting concentration of PTL and MCL was 1 mg/mL.
MCL, resulting in an effective concentration range of the blood
level of MCL for a relatively long time.
The pharmacokinetics of DMAMCL was studied by intraven-
ous injection at 100 mg/kg (Figure 12b), and the pharmacoki-
netic parameters are given in Table 6. The oral bioavailability of
DMAMCL was also determined and found to be 75%. These
data suggest that the basic pharmacokinetic properties of
DMAMCL make it sufficiently promising as a prodrug.
In Vivo Efficacy Study of DMAMCL in the AML NOD/
SCID Murine Model. The potency and selectivity of DMAMCL
were determined in vivo using an AML NOD/SCID murine
G
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■
●
▲
▼
Figure 10. Concentration−time curve of DMAMCL ( ), MCL ( ), DMAPT ( ), and PTL ( ) in HEPES of pH 7.4 (mean SD, n = 4).
■
●
▲
▼
Figure 11. Concentration−time curve of DMAMCL ( ), MCL ( ), DMAPT ( ), and PTL ( ) in mouse plasma (mean SD, n = 4).
◆
■
Figure 12. PK study of a DMAMCL aqueous solution given po administration and iv administration. (a) DMAMCL ( ) and MCL ( ) plasma
◆
■
concentration−time curve in mouse after a single po administration of drug (150 mg/kg) (mean SD, n = 5). (b) DMAMCL ( ) and MCL ( )
plasma concentration−time curve in mouse after a single iv administration of drug (100 mg/kg) (mean SD, n = 5).
DMAPT-treated mouse was 35%, whereas those in the two
DMAMCL-treated animals were 0.005% and 0.12%, indicating
a basically “disease-free” state for these animals at the end of the
study. Furthermore, the average percentage of CD45+CD33+
cells in DMAMCL-treated mice was lower than that of
DMAPT-treated mice (Figure 13a), suggesting that DMAMCL
is superior to DMAPT as a prodrug.
In another experiment, NOD/SCID mice were engrafted with
acute mixed-lineage leukemia cells and then treated with the
same drug treatment protocol as stated above. All animals were
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Table 5. Pharmacokinetic Parameters of DMAMCL and MCL after a Single po Administration of Drug Aqueous Solution (150
mg/kg) (mean SD, n = 5)
param
unit
DMAMCL
SD
MCL
SD
a
AUC (0−t)
ng/mL·h
ng/mL·h
49633.6
50326.8
460496.6
507542.7
8.85
10851.9
10324.7
222944.3
187204.4
2.75
6632.3
6715.2
51360.6
56375.6
7.59
830.1
800.6
17748.2
16132.2
1.77
AUC(0−∞)
AUMC (0−t)
b
AUMC(0−∞)
c
MRT (0−t)
h
MRT(0−∞)
t1/2
h
9.85
1.86
8.28
1.52
h
9.82
5.72
7.92
2.51
Tmax
h
0.19
0.19
0.36
0.20
d
CL
L/h/kg
L/kg
ng/mL
3.08
0.63
22.59
265.27
2607.6
2.6
V
47.38
35.88
108.6
436.7
Cmax
18966.9
3136.5
a
b
c
d
Area under the curve. Area under the first moment of the plasma concentration−time curve. Mean retention time, MRT = AUMC/AUC. Total
body clearance.
Table 6. Pharmacokinetic Parameters of DMAMCL and MCL after a Single iv Administration of Drug Aqueous Solution
(100 mg/kg) (mean SD, n = 5)
param
unit
DMAMCL
SD
MCL
SD
a
AUC (0−t)
ng/mL·h
ng/mL·h
43943.7
44812.3
66198.7
167803.6
1.51
5381.6
6084.5
9444.2
151987.5
0.09
5489.4
5847.3
8135.3
37846.8
1.49
638.2
628.1
1078.1
47828.8
0.15
AUC(0−∞)
AUMC (0−t)
b
AUMC(0−∞)
c
MRT (0−t)
h
MRT(0−∞)
h
3.52
2.57
6.34
7.90
t1/2
h
18.92
7.36
12.66
5.67
d
CL
L/h/kg
L/kg
ng/mL
2.26
0.28
17.25
1.72
V
88.18
56.37
15333
533.45
10712
509.56
1592
Cmax
92901
a
b
c
d
Area under the curve. Area under the first moment of the plasma concentration−time curve. Mean retention time, MRT = AUMC/AUC. Total
body clearance.
+
+
●
Figure 13. In vivo biological activity of DMAPT in a murine model. (a) Percentage of CD45 CD33 cells after treatment with DMAPT ( ) and
■
■
●
DMAMCL ( ). Each or symbol represents a single animal. The mean percentage is indicated by the horizontal bars. (b) Kaplan−Meier survival
curves, control (n = 7), DOX (n = 6), DMAPT (n = 6), DMAMCL (n = 6).
sacrificed in 35 days after the drug treatment, and their bone
marrow was analyzed. The average percentage of the CD45+CD33+
cancer cells in the bone marrow of DMAMCL-treated mice
was 0.14%, which was significantly lower than that of DMAPT-
treated mice (5.7%). Compared to the average percentage of 9.8%
CD45+CD33+ cells in the bone marrow of the saline-treated control
group, there was a 98.6% inhibition of cancer cell engraft for the
DMAMCL group and a 42% inhibition for the DMAPT group
(Table 7). These in vivo experimental data demonstrate that
DMAMCL was superior to DMAPT in efficacy.
Toxicity Evaluation of Compound DMAMCL. As a
preliminary toxicology study, we treated BALB/c mice with
DMAMCL and carried out hematoxylin and eosin (H&E) staining
of the heart, liver, spleen, lung, and kidney. The animals were given
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Table 7. Percentage of CD45+CD33+ Cells in the Bone
Marrow of DMAPT- and DMAMCL-Treated Mice
along with the much higher plasma stability of MCL over
PTL (half-lives of MCL and PTL in mouse plasma, 2.64 vs 0.36,
Table 4), is helpful to control the blood concentration of MCL
within a suitable window, which is important to ensure a high
AUC and to reduce toxicity (Tables 5 and 6). Moreover, MCL
was less toxic to normal cells. In summary, the drug/prodrug
form of MCL/DMAMCL had at least three advantages over
PTL/DMATP: higher stability of active drug, less toxic to normal
cells and normal stem cells, and more sustainable release of active
drug from prodrug. Indeed, 33% of the NOD/SCID mice even
survived in a “disease-free” state at the end of the in vivo efficacy
study engrafted with M5 AML cells; in comparison, all the
DMAPT-treated mice still had a significant percentage of human
primary AML cells remaining (Figure 13). In another similar
in vivo experiment with engrafted acute mixed-lineage leukemia
cells, the percentage of CD45+CD33+ cells in the bone marrow of
DMAMCL-treated mice was significantly lower than that of
DMAPT-treated mice; compared to the control group, this is
98.6% inhibition in the DMAMCL group vs 42% inhibition in the
DMAPT group (Table 7). This indicates that DMAMCL is also a
promising drug candidate for acute mixed-lineage leukemia.
Note that acute mixed-lineage leukemia still has a very poor
prognosis and still needs an optimal therapeutic approach.25 In
addition, the acute toxicity of DMAMCL via oral administration
is very low; the maximum tolerable doses (MTDs) of DMAMCL
are over 1000 mg/kg for a single dose and 500 mg/kg every day
for seven doses, which are 10 times and 5 times the effective dose
(100 mg/kg), respectively. However, the MTD for DMAMCL
via intravenous (iv) administration is around 200 mg/kg for a
single dose (data not shown). This significant difference of MTD
between oral and iv administration may be explained by the
comparison of pharmacokinetic parameters of DMAMCL after iv
and oral administrations (Figure 12). The drug (DMAMCL)
blood concentration over time after iv administration is a “peak”
and “trough” curve, while sustained absorption and release were
observed when DMAMCL was administrated orally. Arglabin,
an anticancer drug administrated by injection and which is
currently in clinical trials in some countries, has a relatively
narrow therapeutic index compared to DMAMCL. The LD50 of
arglabin by intraperitoneal injection is 190−220 mg in mice, and
the MTD of arglabin by iv administration is around the same
range as the effective dose (from 20 to 50 mg/kg daily for 20
days).18 In summary, the significantly broader therapeutic index
is the major advantage of DMAMCL over arglabin, and this result
also suggests that administration routes may have an important
effect on the therapeutic index.
a
DMAPT
group
DMAMCL
group
no.
control
1
2
3
4
5
6
7
8
3.29
1.6
2.87
1.35
36.9
1.91
1.1
0.02
0.056
0.259
0.177
0.141
0.212
0.221
N/A
5.19
1.12
55.1
0.986
1.25
10.8
0.74
0.442
0.373
av
9.8
5.7
42
0.14
98.6
AML cell inhibition compared to the
control (%)
N/A
a
The mice were engrafted with primary mixed-lineage leukemia cells.
1000 mg/kg DMAMCL by gavage in a single dose or 500 mg/kg
every day for 7 days. No pathologic changes were apparent in the
examined tissues (Supporting Information, p S23). This indicates
that DMAMCL is well-tolerated by oral administration.
DISCUSSION
■
Conventional chemotherapies and radiation therapies for cancer
usually affect only differentiated or differentiating cells, which
form the bulk of the tumor. However, in many cases, a small
population of CSCs could remain untouched and cause the
relapse of cancer.1−4 Unfortunately, only a few compounds can
selectively inhibit CSCs and reduce the proportion of CSCs in
vitro or in vivo. Parthenolide, a 10,5-ring structure SL, is able to
target several types of cancer stem cells,9 and its water-soluble
analogue, DMAPT, is currently in clinical trial.9c
We found that some GSLs, 5,7,5-ring structure SLs, and their
derivatives were able to selectively inhibit AML stem and
progenitor cells, including compounds arglabin, 1, 9 (MCL), 10,
12, 19, and 20. The cell viabilities of stem/progenitor cells vs
total leukemia cells after treatment with these compounds were
(arglabin) 10.7% vs 20.0%, (1) 3.8% vs 6.4%, (9) 2.3% vs 8.4%,
(10) 44.5% vs 49.7%, (12) 22.6% vs 30.6%, (19) 2.1% vs 10.8%,
and (20) 11.2% vs 53.1% (Table 2). We also established the first
preliminary SAR against AML stem/progenitor cells (Figure 5).
Note that arglabin is an anticancer drug currently being used for
treatment of breast, liver, and lung cancers in some countries.24
Further investigation demonstrated that MCL, PTL, and arglabin
may selectively kill AML stem cells and then may reduce the
proportion of the AML stem cells in overall surviving AML cells
(Table 3). The targeting of AML stem/progenitor cells with
MCL was confirmed by reduction of the CFUs to the primary
AML cells. Moreover, surviving primary AML cells after MCL
treatment had significantly low engraftment in mice compared to
that of an equal amount of saline-treated primary AML cells
(Figure 9).
DMAPT, the water-soluble dimethylamino Michael adduct of
PTL, was reported to be equally as potent as PTL in vitro,9b while
the Michael adduct of MCL, DMAMCL, was significantly less
active in vitro (Tables 1 and 2). However, in neutral HEPES
solution (pH 7.4) and in plasma, the conversion of DMAMCL to
MCL is much slower than that of DMAPT to PTL (Figures 10
and 11). The preliminary pharmacokinetic (PK) study
demonstrated that DMAMCL showed high oral bioavailability
and constant conversion to active drug MCL. This phenomenon,
CONCLUSION
■
As a speculative view, although the cell-based biology of CSCs
has been studied intensively, small molecules that can selectively
target cancer stem cells are still very rare. The discovery of more
agents that selectively inhibit CSCs is highly urgent for studying
the specific target mechanisms of CSCs. In this context, the
scaffold hopping of the 10,5-ring structure of PTL to the 5,7,5
ring structure of GSLs resulted in the discovery of a library
of anti-AML stem/progenitor cell agents, and the lead com-
pound MCL demonstrates higher plasma stability, more sus-
tained release, and superior in vivo efficacy compared to PTL,
which may be beneficial to in vivo mechanism investigation. This
discovery suggests that GSLs, and probably even all subtypes of
SLs, could be a rich source to identify anti-AML stem/progenitor
cell agents. Indeed, different types of SLs and their derivatives
have been prepared and tested against different types of cancer
J
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BFU-erythrocyte) were scored after 20 days of culture. We counted the
colonies by the colony morphology.
stem/progenitor cells. Moreover, molecular probes based on the
established SAR of GSLs may be developed and will be applied
to identify/confirm their specific target in AML stem cells and
other types of CSCs, especially in vivo. All these works will be
published in due course.
Flow Cytometry. Apoptosis assays were performed as follows. First,
after 18 h of treatment, with the compounds in IMDM, normal and
AML samples were isolated and stained by the antibodies CD34-
allophycocyanin (APC) and CD38-PECy7 for 30 min. The cells were
washed in cold phosphate-buffered saline (PBS) and resuspended in
100 μL of Annexin-V buffer, Annexin-V−fluorescein isothiocyanate
(FITC) and propidium iodide (PI) were added, and the tubes were
vortexed gently. After that the tubes were incubated at room temperature
for 15 min, the samples were analyzed on a BD LSRII flow cytometer
(BD Biosciences). We analyzed the data with Prism 5 software.
NOD/SCID Mouse Assays. NOD/SCID mice were sublethally
irradiated with 280cGay before transplantation. After 6−10 h, the cells
to be assayed (from M5) were injected via tail vein in a final volume of
0.2 mL of PBS. After the cells were transplanted for 8 weeks, DMAMCL
and DMAPT (aqueous solution, 100 mg/kg each time) were administrated
orally every other day. After seven times the administrations were stopped.
Toxicity Evaluation of Compound DMAMCL. Untreated mice
and mice treated with the diluent alone (water) were utilized as controls
to document the normal tissue architecture and inflammation status in
the heart, lung, spleen, and kidney. The experiment was performed with
a total of 16 mice (8 male/8 female) at the indicated time point
examined. Mice were sacrificed at 1 week after DMAMCL (aqueous
solution, 1000 mg/kg for a single dose or 500 mg/kg every day for seven
doses) was orally administered, and the entire tissue was surgically
excised. All the tissues were fixed in 10% neutral-buffered formalin and
embedded in paraffin, and sections of 3 μm thickness were subjected to
H&E staining by standard procedures. Gross morphological analyses
were performed on tissues stained with H&E. Tissue sections from all
mice within a treatment group were visually examined by using an
Olympus IX81 microscope to assess the gross morphological condition.
After the overall assessment of the tissue, a representative field from the
heart, lung, spleen, and kidney of each treatment group was photo-
graphed by using a high-resolution digital camera.
METHODS
■
Synthesis of Compounds 8−22. PTL was dissolved in MeOH/H+
at room temperature and resulted in the formation of compound 8.26
Cyclization of PTL using BF3 in toluene provided compounds 9−12.27
Taking into account that the physiological environment is aqueous, the
cyclization was carried out in an acetone/water mixture to obtain com-
pound 13.28 Compound 14 is a N3 adduct of PTL, and compounds 15−
18 are the products obtained by Cu(I)-catalyzed reaction of compound
14 with various alkynes (Figure 3). MCL (compound 9) can also be
prepared in high yield using our modified method,29 and the derivatives
of micheliolide (compounds 19, 20, 21, and 22) were prepared as
outlined in Figure 4. MCL was treated with MeI and NaH in THF to
provide methylated product 19. Derivative 20 was obtained as a N3
adduct. Compound 21 (DMAMCL) was obtained as a white powder
through dimethylamine addition to MCL,30 followed by addition of 1
equiv of HCl and lipholization. Hydrogenation of MCL with NaBH4
formed dihydromicheliolide (compound 22).
Materials and Experimental Procedure of the PK Study. The
UPLC−MS/MS system consisted of an AcQuity ultraperformance
liquid chromatograph and a Quattro Premier XE mass spectrometer
(Waters/Micromass, Milford, MA). All solvents and chemicals were of
HPLC grade and purchased from Fisher Scientific (Tustin, CA).
Male Kunming mice (20 2 g) were supplied by the lab animal
center of the Academy of Military Medical Science (Beijing, China). The
experimental protocol was approved by the Nankai University Ethics
Committee for the use of experimental animals and conformed to the
Guide for Care and Use of Laboratory Animals. Mice were housed at
22 2 °C and 55 5% relative humidity under a 12 h light−dark cycle.
They were fasted for 12 h before drug administration, and water was
freely available. DMAMCL aqueous solution was po (150 mg/kg) or iv
(100 mg/kg) administered (n = 5). The blood samples were collected
from the orbital veins at the setting time. All the biological specimens
were stored at −80 °C. PK data were generated using noncom-
partmental analysis.
ASSOCIATED CONTENT
■
S
* Supporting Information
Experimental procedure, NMR spectra of compounds 8−22, and
pathology pictures. This material is available free of charge via the
Internet at http://pubs.acs.org/.
Release Studies of Prodrug to Active Drug. A 20 μL volume of
DMAMCL or DMAPT solution was placed in 980 μL of HEPES at
pH 7.4 or mouse blank plasma solution. The tubes were then incubated
in a bath incubator at 37 °C. Samples were removed in 10 s, and the
concentration of DMAMCL, MCL, DMAPT, and PTL was analyzed by
HPLC.
AUTHOR INFORMATION
■
Corresponding Author
*Phone: +86 22 23508090. Fax +86 22 23508090. E-mail:
yuechen@nankai.edu.cn (Y.C.); gaoyingdai@hotmail.com
(Y.G.).
Cell Isolation and Culture. Primary human AML samples were
obtained from volunteer donors from the Institute of Hematology &
Blood Diseases Hospital (Tianjin, China). Umbilical cord blood
samples were obtained from a volunteer donor in the Maternity
Hospital (Tianjin, China). Mononuclear cells were isolated from the
samples using Ficoll-Paque density gradient separation and cryopre-
served in a freezing medium of 90% FBS and 10% dimethyl sulfoxide
(DMSO) until use. Cells were cultured in serum-free IMDM for 1 h
before treatment by the compounds. All the drug treatments were
performed in triplicate. The cell viability assay was carried out using the
well-documented (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazo-
lium bromide (MTT) method. All the tested cells were cultured with
drugs for 72 h before addition of the MTT reagent. All the experiments
were carried out in triplicate, and we tested every compound three times.
Methylcellulose Colony-Forming Assay. The mononuclear cells
from the cord blood of AML patient volunteers were cultured in serum-
free IMDM for 18 h in the presence or absence of drugs. The drug
concentrations were 0.05 μM DOX, 0.10 μM DOX, 5 μM PTL, 10 μM
PTL, 5 μM MCL, and 10 μM MCL, respectively. Cells were plated in
triplicate in the methylcellulose medium M3434 (StemCell Technologies,
Vancouver, Canada), and total colonies (CFC-Mix, CFC-granulocyte
and -monocyte, CFC-granulocyte, CFC-monocyte/macrophage, and
Author Contributions
#These authors contributed equally to this work.
Notes
The authors declare the following competing financial
interest(s): Y.C. is one of the founders of Accendatech Co.,
Ltd., which has a financial interest in DMAMCL. All other
authors declare no competing financial interests.
ACKNOWLEDGMENTS
■
This work was supported by the National Basic Research Pro-
gram of China (Grants 2011CB964801 and 2012CB966604),
the National Natural Science Foundation of China (NSFC)
(Grants 21072106 to Y.C., 81001377 to Q.Z., 90913018,
81090410, and 81170465), The Ministry of Science and
Technology (Grant 2009CB918901), the Fok Ying Tong
Education Foundation (Grant 122037), and The Natural Science
Foundation of Tianjin (TJNSF) (Grant 09JCZDJC21900).
K
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ABBREVIATIONS USED
■
CSC, cancer stem cell; PTL, parthenolide; SL, sesquiterpene
lactone; SERCA, sarco/endoplasmic reticulum calcium transport
ATPase; LSC, leukemia stem cell; BCSC, breast cancer
stem cell; GSL, guaianolide sesquiterpene lactone; DMAPT,
(dimethylamino)parthenolide; MCL, micheliolide; DMAMCL,
(dimethylamino)micheliolide; NOD/SCID, nonobese diabetic/
severe combined immunodeficiency; DOX, doxorubicin; HSC,
hematopoietic stem/progenitor cell; CFU, colony-forming unit;
AUMC, area under the first moment of the plasma
concentration−time curve; MRT, mean retention time, MRT =
AUMC/AUC; CL, total body clearance
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