Synthesis and evaluation of bisbenzylidenedioxotetrahydrothiopranones as activators of endoplasmic reticulum (ER) stress signaling pathways and apoptotic cell death in acute promyelocytic leukemic cells

Article abstract of DOI:10.1021/jm401352a

Curcumin is known to trigger ER-stress induced cell death of acute promyelocytic leukemic (APL) cells by intercepting the degradation of nuclear co-repressor (N-CoR) protein which has a key role in the pathogenesis of APL. Replacing the heptadienedione mo

Full text of DOI:10.1021/jm401352a

Article
pubs.acs.org/jmc
Synthesis and Evaluation of Bisbenzylidenedioxotetrahydrothiopranones
as Activators of Endoplasmic Reticulum (ER) Stress Signaling Pathways
and Apoptotic Cell Death in Acute Promyelocytic Leukemic Cells
Kheng-Lin Tan,† Azhar Ali,‡ Yuhong Du,§ Hainan Fu,§ Hai-Xiao Jin,∥ Tan-Min Chin,‡ Matiullah Khan,⊥
and Mei-Lin Go*,†
†Department of Pharmacy, National University of Singapore, 18 Science Drive 4, 117543, Republic of Singapore
‡Cancer Science Institute of Singapore, Yong Loo Lin School of Medicine, National University of Singapore, 10 Medical Drive,
117597, Republic of Singapore
§Department of Pharmacology, Emory Chemical Biology Discovery Centre, Emory University School of Medicine,
1462 Clifton Road, Atlanta, Georgia 30322, United States
∥Key Laboratory of Applied Marine Biotechnology, Ministry of Education, School of Marine Sciences, Ningbo University,
Fenghua Road 818, Jiangbei District, Ningbo, Zhejiang 315211, People’s Republic of China
⊥School of Medicine, Asian Institute of Medicine, Science and Technology (AIMST), Jalan Bedong Semeling, 08100, Bedong,
Kedah, Malaysia
S
* Supporting Information
ABSTRACT: Curcumin is known to trigger ER-stress induced cell death of
acute promyelocytic leukemic (APL) cells by intercepting the degradation of
nuclear co-repressor (N-CoR) protein which has a key role in the pathogenesis
of APL. Replacing the heptadienedione moiety of curcumin with a
monocarbonyl cross-conjugated dienone embedded in a tetrahydrothiopyr-
anone dioxide ring resulted in thiopyranone dioxides that were more resilient
to hydrolysis and had greater growth inhibitory activities than curcumin on
APL cells. Several members intercepted the degradation of misfolded N-CoR
and triggered the signaling cascade in the unfolded protein response (UPR)
which led to apoptotic cell death. Microarray analysis showed that genes
involved in protein processing pathways that were germane to the activation of
the UPR were preferentially up-regulated in treated APL cells, supporting the
notion that the UPR was a consequential mechanistic pathway affected by
thiopyranone dioxides. The Michael acceptor reactivity of the scaffold may
have a role in exacerbating ER stress in APL cells.
ER-associated degradation (ERAD) of misfolded N-CoR.12−14
If ER stress persists, the UPR shifts to a prodeath mode in an
1. INTRODUCTION
Acute promyelocytic leukemia (APL) is caused by the fusion
effort to eliminate cells harboring deleterious malfolded
protein PML-RARα that is formed by the reciprocal trans-
proteins.10,11,15 Curcumin, the active substance of the spice
location of PML and RARα genes located at chromosomes 15
and 17.1,2 The oncogenic potential of the PML-RARα fusion
turmeric (Curcuma longa), was reported to block the protease-
and proteasomal-mediated degradation of N-CoR.16 The
protein is linked to the nuclear receptor co-repressor (N-CoR)
protein,3−6 which is recruited by the fusion protein to give the
resulting build-up of misfolded N-CoR amplified ER stress
and caused the UPR to switch from a prosurvival to a
proapoptotic mode. ER stress-induced apoptosis is increasingly
N-CoR-PML-RARα complex. The latter represses retinoic acid
responsive genes that are essential for the maturation of
promyelocytic cells, hence curtailing cell differentiation and
viewed as an attractive and novel signaling target for anticancer
therapies,11,17 and the ability of curcumin to sensitize APL16
and other malignant cells18−21 to apoptosis underscores its
anticancer potential. There are however several shortcomings to
curcumin that has limited its therapeutic usefulness, notably its
modest potency, intrinsic instability, and limited bioavailability.22,23
transformation.7,8 The PML-RARα fusion protein also induces
misfolding of N-CoR which leads to increased levels of
endoplasmic reticulum (ER) stress due to the accumulation of
misfolded protein.9 In an effort to restore ER homeostasis, cells
activate the unfolded protein response (UPR) which entails
temporarily shutting down protein translation and up-
regulating ER folding and quality control mechanisms.10,11 In
APL cells, this is achieved in part by protease-mediated and
Received: September 3, 2013
© XXXX American Chemical Society
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Figure 1. Structures of curcumin and representative analogues designed to address the activity and pharmacokinetic limitations of curcumin.25,26
Figure 2. Replacement of the β-diketone moiety of curcumin by monocarbonyl cross-conjugated dienones (monocarbonyls, cyclopentanones,
cyclohexanones, thiopyranones, piperidinones). The thiopyranones 32, 34, and 39 had potent growth inhibitory activities (IC50) on NB4, an APL
cell line,27 and were targeted for further modifications (shown in box) in this report.
Various strategies have been proposed to address these
limitations such as engaging novel formulations to enhance
delivery of curcumin22,24 and modifying the curcumin scaffold
to improve pharmacokinetic and potency end points.25,26 Some
examples are provided in Figure 1. We had previously
investigated the APL growth inhibitory activity of compounds
derived by replacing the conjugated β-diketone moiety of
curcumin with a monocarbonyl cross-conjugated dienone that
was either unmodified (“monocarbonyls”) or embedded in
carbocyclic (“cyclopentanones”, “cyclohexanones”) or heterocyclic
(“thiopyranones”, “piperidinones”) rings (Figure 2).27 Several
compounds bearing the tetrahydrothiopyran ring (“thiopyranones”)
were found to arrest APL cell proliferation at lower concentrations
(IC50 = 0.3−1 μM) than curcumin (IC50 = 5.5 μM). To further
explore the potential of this scaffold, additional structural
modifications were investigated in this report, namely, asym-
metrical substitution of the terminal phenyl rings, switching of
the latter to pyridine, and replacement of the thiopyranone ring
with thiopyranone 1,1-dioxide (Figure 2). Herein we describe the
synthesis and growth inhibitory properties of these compounds
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on APL cells, the hydrolytic stabilities of selected members, and
in view of their structural connection to curcumin, the
involvement of ER stress-induced apoptosis in the mechanistic
pathways of potent members. In addition, the effects of a
representative potent member (41) on the expression of genes
involved in ER protein processing and on selected proteins/
transcription factors that mediate the response to ER stress in the
UPR signaling pathway were examined for mechanistic insight.
was dehydrated in the presence of p-toluenesulfonic acid to give 2
(21% isolated yield). Acid-catalyzed aldol condensation of 2 and
the methoxy or hydroxyl substituted benzaldehyde gave the
desired thiopyranones 217−220.
2.2. Growth Inhibitory Activities of Curcumin Ana-
logues on APL Cell Lines. The test compounds were
evaluated on two APL cell lines, NB4 and NB4-R1. NB4 cells
respond to retinoic acid which is the standard treatment for
APL,32 whereas NB4-R1, a de novo cell line derived from
parental NB4 cells,33 is retinoic acid resistant. Table 1 lists the
growth inhibitory IC50 values (concentration required to reduce
cell viability to 50% of levels observed in untreated cells after
72 h of exposure) of the test compounds on these cell lines. Also
included in Table 1 are previously determined IC50 values of
selected thiopyranones, cyclohexanones, and piperidinones.27
2. RESULTS
2.1. Chemistry. In a previous report, thiopyranones 32, 34,
39 (Figure 2) were found to be potent inhibitors of APL cell
viability.27 Their antiproliferative activities were broadly
comparable to their piperidinone counterparts but exceeded
that of the monocarbonyl, cyclopentanone, and cyclohexanone
analogues. Unlike the piperidinones which have been
intensively investigated for their anticancer and anti-inflamma-
tory activities,28−31 less is known of the structure−activity
relationship or mechanistic profile of thiopyranones. Hence, to
appropriate the full potential of this scaffold, we undertook the
following modifications. First, fluoro, hydroxyl, and/or methoxy
groups were introduced at various positions on the terminal
rings. As shown in Figure 2, these groups were present in the
earlier hits 32, 34, and 39.27 Second, asymmetric substitution of
the phenyl rings was explored with the above-mentioned
groups. Third, the phenyl rings were replaced by pyridines,
motivated in part by the good results observed for several
cyclohexanones27 and piperidinones28 which had similar
phenyl-to-pyridine substitution. Lastly, the thiopyranone ring
was replaced by thiopyranone 1,1-dioxide. Table 1 lists the
compounds synthesized in this report. Besides the thio-
pyranones (series I) and thiopyranone dioxides (series II), several
cyclohexanones (series III) and piperidinones (series IV) were also
prepared for comparison to their sulfur-containing counterparts in
series I and II.
Series I, III, and IV compounds that have symmetrically
substituted phenyl rings were obtained in good yields by a base-
catalyzed aldol condensation reaction between the ketone
(cyclohexanone, tetrahydrothiopyranone, piperidinone) and an
arylcarboxaldehyde (Scheme 1A). In the case of benzaldehydes
with phenolic hydroxyl groups, no hydroxyl protection was
required when aldol condensation with the ketone (tetrahy-
drothiopyranone, piperidinone) was carried out under acidic
conditions. The final products (215, 216, 230, 231) were
obtained in reasonably good yields (24−60%). Oxidation of the
thiopyranone sulfide with 30% hydrogen peroxide in acetic acid
gave the corresponding thiopyranone 1,1-dioxides of series II
(Scheme 1A).
The most potent thiopyranones in Table 1 were 207 (IC50
=
0.17 μM) and 208 (IC50 = 0.11 μM) which have pyridine in
place of phenyl as terminal rings. Their growth inhibitory
activities surpassed that of 32 (IC50 = 0.29 μM) which was the
most potent thiopyranone identified earlier.27 Interestingly, the
growth inhibitory activities of 207 and 208 were comparable to
that of the potent cyclohexanones 24 (0.19 μM) and 25 (IC50 =
0.22 μM) which also have terminal pyridines. Furthermore, a
recent report highlighted potent kinase inhibition by a
piperidinone analogue with terminal 2-pyridinyl rings.28 Thus,
there is anecdotal support for the activity-enhancing potential
of a phenyl to pyridine substitution across various carbonyl
cross-conjugated dienone scaffolds related to curcumin.
A comparison of the growth inhibitory activities of
thiopyranones and cyclohexanones revealed interesting scaf-
fold-dependent differences in the structure−activity relation-
ship (SAR). Notably, a regioisomeric preference was observed
among the pyridine-bearing thiopyranones that was not
apparent among the cyclohexanone counterparts (24−26).
For instance, thiopyranone 209 with 4-pyridinyl rings was less
active than its 2- or 3-pyridinyl counterparts, a distinction that
was not seen among the pyridine-bearing cyclohexanones.
Additionally, methoxy substitution of the pyridine ring was well
tolerated in the thiopyranone 210 (IC50 = 0.17 μM versus
unsubstituted 208 IC50 = 0.11 μM) but not in the cyclohexanone
201 (IC50 = 1.06 μM versus unsubstituted 25 IC50 = 0.22 μM).
The presence of hydroxyl/methoxy groups in the hit
compounds 34 and 39 prompted us to investigate more of
such thiopyranones (211, 213−216), but disappointingly, only
incremental improvements in activity were observed. Notably,
the most promising thiopyranone 215 (R = 3-OH, 4-OCH3;
IC50 = 0.38 μM) was marginally more active than the original
hits 34 and 39. On the other hand, dimethoxy and trimethoxy
substitution resulted in exceptionally poor activities as seen
from 213 (2,5-OCH3, IC50 = 3.23 μM) and 214 (3,4,5-OCH3,
IC50 = 3.82 μM). The contrasting activities of 213 and its
3,4-regioisomer 39 were striking and pointed to a preference
for certain substitution patterns on the terminal rings. Again,
scaffold-dependent SAR was apparent as piperidinones bearing
the same dimethoxy/trimethoxy (228, 229) groups retained
outstanding submicromolar activities. In fact, the trimethoxy
substituted piperidinone 229 (IC50 = 0.10 μM) was the most
potent member in its class.
The asymmetrically substituted cyclohexanones (202−205)
and thiopyranones (217−220) were prepared sequentially by
reacting the ketone with 3-fluorobenzaldehyde to give inter-
mediates (1, 2) which were then reacted in a second aldol
condensation under acidic conditions with the methoxy/hydroxyl
substituted benzaldehyde. The intermediate 2-(3-fluorobenzyl-
idene)cyclohexanone (1) was obtained under acidic conditions in
the presence of a large excess of cyclohexanone (Scheme 1B), but
strong basic conditions were required for the synthesis of
intermediate 2 ((3-fluorobenzylidene)dihydro-2H-thiopyran-
4(3H)-one, Scheme 1C). To obtain 2, dihydro-2H-thiopyran-
4(3H)-one was reacted with lithium diisopropylamide (LDA) at
low temperatures to generate the nucleophilic enolate which
attacked the electrophilic carbonyl carbon of 3-fluorobenzaldehyde
to give the hydroxyl intermediate on aqueous workup. The latter
For thiopyranones with asymmetrically substituted phenyl
rings in which 3-fluoro was retained on one phenyl ring and
hydroxyl/methoxy groups on the other ring, no apparent
advantage was observed among the asymmetrically substituted
̀
analogues (217−220) vis-a-vis their symmetrically substituted
C
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Table 1. Mean Growth Inhibitory Activity (IC50, μM) of Compounds on Two Human APL Cell Lines
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Table 1. continued
a
b
Concentration required to effect 50% inhibition of cell growth. Results are the mean standard deviation of at least three independent experiments. R1 =
c
d
R2 unless indicated otherwise. APL cell lines. NB4 is sensitive to retinoic acid. NB4-R1 is resistant to retinoic acid. IC50 values were previously
reported.27 Not determined. IC50 not determined above 5 μM. Phenyl replaced by pyridine (Py). IC50 not determined above 40 μM because of
e
f
g
h
solubility issues.
counterparts (32, 34, 39, 215). A similar trend was observed
for the asymmetrically substituted cyclohexanones (202−205).
Oxidation of the thiopyranone sulfide to sulfone gave rise to
a series of thiopyranone dioxides (40, 41, 221−227). Here, we
E
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a
Scheme 1
a
(A) Synthesis of selected series I−III analogues, reagents and conditions: (a) substituted benzaldehyde or pyridinecarboxaldehyde (2.1 equiv),
NaOH (3 equiv), EtOH, 25 °C, 18−24 h; (b) substituted benzaldehyde (2.1 equiv), conc HCl, EtOH, 60 °C, 18−24 h; (c) For X = S: 30% H2O2,
CH3COOH, 60 °C, 6−24 h. (B) Synthesis of asymmetrical series III analogues 203−205, reagents and conditions: (a) 3-fluorobenzaldehyde (2.1
equiv), conc HCl, EtOH, 60 °C, 18−24 h; (b) substitituted benzaldehyde (2.1 equiv), conc HCl, EtOH, 60 °C, 18−24 h. (C) Synthesis of
asymmetrical series I analogues 217−220, reagents and conditions: (a) LDA (2 M), dry THF, −78 °C, 1 h; (b) 3-fluorobenzaldehyde (1 equiv), dry
THF, −78 °C, 6 h; (c) p-TsOH (5 mol %), toluene, 90 °C, 1.5 h; (d) substituted benzaldehyde (1 equiv), conc HCl, EtOH, 60 °C,
18−24 h.
found potent nanomolar activities among the fluoro substituted
analogues 40, 41, 221 (IC50 = 0.05−0.08 μM), the trimethoxy
analogue 224 (IC50 = 0.05 μM), and the 3-fluoro-4-methoxy
analogue 227 (IC50 = 0.08 μM). When compared with
cyclohexanones, thiopyranones, and piperidinones bearing
similar ring substituents, the thiopyranone dioxide analogue
was clearly superior in terms of growth inhibitory activity.
Notably, 41 and 224 were nearly twice as potent as their
piperidinone counterparts (43 and 229), and 227 far exceeded
cyclohexanone 206 and thiopyranone 211 (IC50 ≈ 5 μM)
which have the same ring substituents.
We then considered the antiproliferative activities of the test
compounds on the retinoic acid resistant NB4-R1 cell line.
Curcumin was equipotent on both NB4 (IC50 = 6 μM) and
NB4-R1 (IC50 = 8 μM) cells, and this was also observed for
most of the compounds listed in Table 1. A notable exception
was the trimethoxy-substituted thiopyranone 214, which was
at least 20 times more potent on NB4-R1 (IC50 = 0.16 μM)
than NB4 (IC50 = 3.82 μM). Otherwise, SAR trends were
generally similar to those observed on the retinoic acid sensitive
NB4 cells.
2.3. Hydrolytic Stabilities of Thiopyranone Dioxides
41 and 227 and Thiopyranone 219. Liang and co-workers
reported that replacing the conjugated diketone linker of
curcumin with a monocarbonyl dienone moiety resulted in
analogues with improved hydrolytic stabilities.34 They inves-
tigated compounds that were structurally related, but not
identical, to those investigated in this report and found that
generally for the same substitution on the terminal phenyl
rings, stabilities were of order cyclopentanones (most stable) >
cyclohexanones > monocarbonyls (least stable). As thiopyr-
anones and thiopyranone dioxides had shown good growth
inhibitory activities (Table 1), we proceeded to assess their
degradation of curcumin (>90% after 75 h). Thiopyranone 219
and thiopyranone dioxides 41, 227 were hydrolytically more
stable but degraded over time, albeit to a lesser extent than
curcumin. Thiopyranone dioxide 227 was initially more
resistant to hydrolysis than 41 and 219 but after 75 h, all 3
compounds had broadly comparable levels of degradation
(60−68%) (Supporting Information, Figure S1)
2.4. Thiopyranone Dioxide 41 and Related Com-
pounds (219, 227) Induce Apoptotic Cell Death in NB4
Cells. Next, we went on to determine if the potent analogues
41, 219, and 227 induced apoptotic cell death. The compounds
were incubated with NB4 cells for 48 h at their IC50 NB4 and
higher concentrations, after which levels of cleaved caspases 3
and 9 and PARP were probed by Western blotting (Supporting
Information, Figure S2). 41 and 219 induced dose dependent
increases in cleaved caspase 9 levels at concentrations ranging
from 1 × IC50 to 5 × IC50. In contrast, 227 did not activate
caspase 9 even at 10 × IC50. As cleavage of caspase 9 is initiated
by the release of cytochrome c from the mitochondria in the
intrinsic apoptotic pathway, this pathway may not be involved
in 227-induced apoptosis. All three compounds promoted the
cleavage of the downstream executioner caspase 3 which was
common to both intrinsic and extrinsic apoptotic pathways.
Levels of cleaved PARP were also elevated. The induction of
apoptotic cell death was further confirmed for 41 and 219 by
FACS analysis of NB4 cells double stained with annexin V and
propidium iodide (Figure S2).
2.5. Thiopyranone Dioxide 41 Induce Accumulation
of N-CoR Protein in NB4 Cells. The accumulation of
misfolded N-CoR is an incipient event leading to ER stress
amplification and NB4 cell death.9,12 Corroboratory evidence
was adduced from curcumin which sensitized NB4 cells to ER
stress induced apoptosis by blocking the loss of misfolded
N-CoR16 and structurally related monocarbonyl dienone
analogues where accumulation of misfolded and nonfunctional
N-CoR was cited as a basis of selective activity on NB4 cells.27
̀
hydrolytic stabilities vis-a-vis curcumin. Hence, the stabilities of
representative members (41, 219, 227) were determined at
pH 7.4, 25 °C over 75 h. We found substantial time-dependent
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Figure 3. Thiopyranone dioxide 41 induced accumulation of misfolded N-CoR in NB4 cells. NB4 cells were treated for 48 h with test compound.
(A) N-CoR protein levels in whole cell extracts were probed by Western blotting using anti-N-CoR antibody. Protein loading in each lane was
monitored with coomassie stain. 41 increased N-CoR levels at 2.5, 5.0, and 25 nM. (B) Vehicle control comprises media with 0.5% DMSO. Cells
were stained with DAPI to visualize cell nucleus (blue, left panel) and anti-N-CoR antibody to visualize N-CoR (red, middle panel). Localization of
N-CoR in cytosol was shown by red ring around a blue core. Cytosolic N-CoR was detected in cells treated with 41 at 2.5 nM to 25 nM.
Experiments were repeated at least twice for each compound at stated concentrations.
Having shown that thiopyranone dioxide 41 potently induced
apoptosis, we proceeded to assess the involvement of N-CoR in
the death process. NB4 cells were treated with 41 for 48 h, after
which levels of N-CoR were probed by Western blotting.
Control cells showed a faint band corresponding to low basal
levels of full length N-CoR (Figure 3A) which corroborated
earlier findings that N-CoR was predominantly fragmented
in NB4 cells, with only a small proportion present in the
intact folded or misfolded state which constituted full-length
N-CoR.12 Treatment with 41 at 5 nM intensified this band but
with poor dose dependency. For further confirmation, we
turned to in vitro immunofluorescence imaging to determine if
the full length N-CoR was localized in the nucleus or cytosol.
Only natively folded N-CoR was localized in the nucleus,
whereas misfolded N-CoR was predominantly cytosolic.12 In
this method, N-CoR was detected by its antibody which gave a
red fluorescence. When present in the nucleus, N-CoR had a
purplish hue due to the merging of blue DAPI and red N-CoR
images. Figure 3B shows muted red fluorescence in control
NB4 cells, in keeping with low basal levels of N-CoR in these
cells. When exposed to 41 (2.5 nM to 25 nM), the red N-CoR
fluorescence intensified, signifying an increase in N-CoR levels.
The increases were localized in the cytosol as seen from the red
rings that surrounded the DAPI-stained (blue) nuclei (merged
panel) which implied that most of the accumulated N-CoR
induced by 41 was cytosolic and misfolded.
nonstressed situations by chaperone proteins. In the presence
of ER stress, these chaperones are released from the ER stress
sensors for binding to misfolded proteins. The liberated ER
stress sensors would then trigger UPR signaling pathways.
Thus, levels of ER stress chaperone proteins PDI, GRP78, and
HSP90 were probed by Western blotting in NB4 cells treated
with 41, 219, and 227 (Supporting Information, Figure S3). All
three compounds increased PDI, GRP78, and HSP60 levels,
with 41 more potent than 219 and 227. In the case of 41,
increases were evident at 250 nM compared to 2.5 μM (219)
and 1 μM (227) respectively (Figure S3A).
Having shown that 41, 219, and 227 amplified ER stress in
NB4 cells, we proceeded to investigate their effects on the ER
stress sensors that mediate UPR signaling. These sensors
(PERK, IRE1, and ATF6) are involved in both prosurvival and
prodeath functions.10,11,15 The switch from the initial
prosurvival to the late prodeath response is determined in
part by the relative stabilities of the mRNAs/proteins involved
in the signaling pathways and the persistence of the ER stress.10
Here, we determined levels of selected proteins recruited by
PERK and IRE1 in NB4 cells treated with 41, 219, and 227
at their growth inhibitory IC50 NB4 for 24 and 48 h. PERK is
activated early in the UPR and blocks general protein synthesis
by phosphorylating elF2α. Persistent ER stress would direct
signaling along the PERK-elF2α-ATF4 arm toward apoptotic
cell death. Commitment to apoptosis is also relayed by dephos-
phorylation of elF2α by GADD34, a proapoptotic protein,
thereby reversing the elF2α-induced block on translation.
Time-dependent increases in phosphorylated PERK, phosphory-
lated elF2α, and GADD34 were observed in treated NB4 cells
(Figure S3B).
2.6. Thiopyranone Dioxide 41 and Related Com-
pounds (219, 227) Activate the Unfolded Protein
Response (UPR) in NB4 Cells. Next, we queried if the
accumulation of misfolded N-CoR by 41 would amplify ER
stress in NB4 cells. The UPR signaling pathway is mediated by
ER stress sensors which are maintained in inactive states in
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Figure 4. Validation of selected genes that were up- or down-regulated by 41 (50 nM) or curcumin (0.5 μM) by quantitative real-time PCR analysis.
NB4 cells were treated with vehicle (0.1% DMSO), curcumin (5 μM), or 41 (50 nM) for 48 h. Real-time PCR was carried out to measure gene
expression levels in treated samples. Changes in expression levels were compared to levels in control cells (normalized to 1) and presented as mean
fold change relative to control. Results are the mean and SD of three independent experiments.
The IRE1 pathway is the final arm of the UPR and plays an
important role in initiating proapoptotic signals. Initially, IRE1
aids cell survival by up-regulating the expression of ER stress
chaperone proteins and genes involved in protein degradation,
but if ER stress persists, IRE1 triggers apoptosis by recruiting
ASK1 and JNK. Here, we monitored ASK1 and JNK (p54,
p46) levels and observed increases in the phosphorylated states
of both proteins in 219- and 227-treated cells (Figure S3C).
We noted that 41 up-regulated phosphorylated ASK1 but
did not increase phosphorylated JNK at the time points
investigated. Taken together, we have shown that the test
compounds activated ER chaperone proteins, caused sustained
signaling in the PERK and IRE1 arms of the UPR, and activated
downstream molecules (GADD34, ASK1, JNK) that were
involved in relaying the proapoptotic signals to the final
execution phase.
2.7. Thiopyranone Dioxide 41 Induces Expression of
Genes Involved in Protein Processing in the Endoplas-
mic Reticulum. Curcumin is known to act on several protein
targets,25,35 and 41 which is structurally related to curcumin
may be similarly disposed to a pleiotropic profile. If the latter is
true, we would then have to ask if ER stress and UPR signaling
are the main triggers of 41-induced apoptotic cell death which
is central to our hypothesis. Hence, we monitored the gene
expression profile of NB4 cells treated with 41 at its IC50 NB4
concentration of 50 nM for 48 h. Analysis of the results
revealed that 599 genes were significantly altered by 2-fold or
more (adjusted p value of <0.05) in 41-treated cells, with most
genes (581) up-regulated. KEGG pathway analysis of the
microarray profiles identified three key pathways that harbored
the largest number of up-regulated genes, namely, those
involved in metabolism (26 genes), protein processing in the
ER (22 genes), and RNA transport (21 genes) (Supporting
Information, Table S1). Genes that were up-regulated in the ER
protein processing pathway had roles in ubiquitin attachment,
protein transport, folding, and synthesis. SEC63, which plays a
role in post-translational protein translocation in ER, was up-
regulated to the greatest extent by 41 (48× compared to
untreated cells).
by curcumin were also those identified for 41 (Table S1). In
the ER protein processing pathway, 21 common genes were up-
regulated to almost the same levels as 41. SEC63 was also the
most highly up-regulated gene in curcumin-treated NB4 cells.
Only a handful of genes were down-regulated by 41 (18) and
curcumin (12). Of these, six genes were down-regulated by
both compounds (Table S1), and they were involved in cell
development and proliferation (OCM, RGL2, CFB), protein
translation (NXF2, MRPL36), and DNA-binding transcrip-
tional repression (THAP5).36,37
To validate the microarray results, we determined the
expression of randomly selected genes by real-time PCR. These
were UBE2E1, EIF2AK2, SEC62, LMAN1 which were up-
regulated by both compounds in the ER protein processing
pathway and the DNA-binding transcriptional repressor
THAP5 which was down-regulated. Figure 4 shows that 41
and curcumin affected the expression of these genes as
predicted from the microarray results. Although the fold-
changes induced by 41 and curcumin were broadly comparable
(2- to 3-fold), they were elicited at different concentrations
(50 nM 41 versus 5 μM curcumin).
2.8. Michael Acceptor Reactivities of Thiopyranone
Dioxides 41 and 227. Michael adduct formation has been
implicated in the induction of ER stress.38 Briefly, Michael
acceptors react with cysteine residues on secretory proteins and
disrupt the normal sequence of disulfide bond formation
necessary for protein folding. As the load of misfolded protein
increases, ER stress is exacerbated. Thus far, we have reasoned
that the thiopyranone dioxides sensitized NB4 cells to ER stress
induced apoptosis by promoting levels of misfolded N-CoR.
It is conceivable that the Michael acceptor reactivities of the
α,β-unsaturated dienone motif in these compounds may inter-
cept the folding process of proteins (other than or including
N-CoR). Hence, it was of interest to assess the Michael acceptor
reactivities of these compounds. Here, we employed a proton
NMR spectroscopic method to monitor the reaction of 41, 227,
and curcumin with cysteamine, a nucleophile with an affinity
for Michael acceptors.39 Briefly, the spectrum of the test
compound in deuterated DMSO was first recorded, after which
a 2-fold excess of cysteamine was added and the spectrum
recorded again after 5 min. The reaction of curcumin with
cysteamine had been reported by this method,39 and our
The gene expression profile of curcumin-treated NB4 cells
was also investigated under similar conditions but at a higher
concentration (5 μM). Interestingly, the key pathways affected
H
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Table 2. Concentration of Test Compound (41, Curcumin, Bortezomib) Required To Inhibit Catalytic Activity of Human 26S
Proteasomal Subunits by 50% (IC50) after 2 and 4 h, 25°C
a
IC50 (nM)
41
curcumin
bortezomib
b
b
b
human 26S proteasomal catalytic subunit
2 h
4 h
1900 200
3800
5400 900
2 h
4 h
2 h
4 h
trypsin-like
26000 2000
40000 4000
26000 1000
2500 200
4800 700
3800 800
5.3 0.4
6.6 0.7
2.7 0.3
1.9 0.2
1.2 0.3
1.0 0.0
caspase-like
26000
0
0
chymotrypsin-like
45000 5000
48000
0
a
b
Mean and SD for n = 3 separate determinations. Basal activity of catalytic subunits at 4 h was approximately 80% of basal activity levels at 2 h.
Figure 5. Orientation of (A) 41, (B) 227, and (C) curcumin in the binding pocket of β5 subunit of the 20S proteasome (PDB 1IRU).41 The
binding pocket is “T” shaped with lipophilic (green), H bonding (magenta), and mildly polar (blue) areas (MOE, version 11). 41 (A) and 227 (B)
were oriented along the horizontal arm of the T, and their phenyl rings established π−π/hydrophobic interactions with the lipophilic residues at the
ends of the horizontal arm. These were Tyr 169 on one end and Ala20, Ala49, Val31, Met45 on the other end. (C) Curcumin had an “L-shaped”
orientation and occupied part of the horizontal and vertical axes. The vertical arm comprised a limited hydrophobic region at the base of the T. The
enolic OH was H-bonded to Thr1 and Ser130, and one of its phenyl rings established nonpolar interactions with the hydrophobic domain (Val128,
Ala46, Met97) at the base of the vertical axis. (D) Hydrogen bonding and π−π and nonpolar interactions of 41 with binding pocket.
findings showed a loss of olefinic protons (Hα, Hβ) and
appearance of multiple peaks in the region 6.50−7.50 ppm
(due to adduct formation with cysteamine) as reported earlier
(Supporting Information, Figure S4).39 Because of the sym-
metrical nature of 41 and 219, the two olefinic protons Hα
were observed as a single peak at 7.85 ppm (41) and 7.80 ppm
(227). On addition of cysteamine, the spectra showed
significant reduction in peak intensities, changes in chemical
shifts, and the appearance of multiple peaks in the region 6.90−
8.00 ppm that were indicative of Michael adduct formation
(Supporting Information, Figure S4).
2.9. Thiopyranone Dioxides 41, 221, and 227 Inhibit
26S Proteasomal Activity in Vitro. Curcumin was reported
to promote accumulation of misfolded N-CoR by blocking its
degradation by N-CoR specific proteases and the proteasome.16
O-Sialoglycoprotein endopeptidase (OSGEP), a protease selec-
tively expressed in APL cells to cleave misfolded N-CoR,12 was
inhibited by curcumin but not 41 (data not shown). Thus, 41
may intercept the degradation of misfolded N-CoR by other
means, possibly by inhibiting the proteasome. To investigate
this possibility, we evaluated 41 for inhibition of purified 26S
proteasomal subunits (chymotrypsin-like, trypsin-like, caspase-
like). Briefly, the proteasome was incubated with various
concentrations of 41 for 2−4 h at 25 °C, after which residual
proteasomal activity was evaluated with luminogenic peptide
substrates that were specifically cleaved by the different catalytic
subunits. 41 was found to inhibit trypsin, caspase, and
chymotrypsin-like activities to the same extent, based on IC50
values (Table 2). Inhibitory potencies were comparable to
curcumin (a known proteasomal inhibitor)40 but significantly
weaker than the positive control bortezomib. We noted that
inhibition increased with incubation time and in the case of 41,
lengthening the incubation time from 2 to 4 h led to a decrease
in IC50 values from 26−45 μM to 2−5 μM. Less pronounced
decreases were observed for bortezomib under similar
conditions.
To determine the structural determinants of 41 that could
have contributed to proteasomal inhibition, we docked 41
onto the β5 subunit (chymotrypsin-like activity) of the human
20S proteasome (PDB 1IRU).41 The binding pocket had a
skewed “T” shape, with lipophilic residues at both ends of the
horizontal arm of the T (Tyr169, Ala20, Ala49, Val31, Met45).
Sandwiched between these lipophilic domains was a cluster of
H bonding residues (Thr1, Thr21, Ser130, Lys33). Inspection
of the top ranked poses of 41 showed that it was aligned along
the horizontal arm of the T shaped pocket, with one terminal
phenyl ring involved in π−π stacking with Tyr 169 and the
other flanked by nonpolar residues Ala49, Val31, and Met45.
The sulfonyl oxygen atoms were engaged in H bonding with
Thr1 (N-terminal NH2) and Thr21 (backbone NH) (Figure 5A
and Figure 5D). We found similar poses for two other
thiopyranone dioxides 221 and 227 (Figure 5B), which suggested
that the scaffold was well placed for productive H bonding and
π−π and hydrophobic interactions with the β5 binding pocket.
Curcumin was also accommodated in the binding pocket, but
unlike 41, it had an L-shaped pose and occupied the vertical arm
and half of the horizontal T arm (Figure 5C).
Next, we assessed 41 for inhibition of the proteasome in NB4
cells. Briefly, 41 was incubated with NB4 cells (48 h, 37 °C),
after which residual proteasomal activities were determined
with fluorogenic peptide substrates specific to the catalytic
subunits. 41 was found to completely inhibit caspase-like,
trypsin-like, and chymotrypsin-like activities at 1 μM
(Supporting Information, Figure S5) which was not in keeping
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with its modest in vitro inhibitory activities (IC50 = 2−5 μM)
observed earlier. The different experimental conditions
employed in these assays may have contributed to the contrast-
ing results, but the possibility of 41 inhibiting other cellular
proteases that could hydrolyze the fluorogenic substrates
should not be entirely discounted.
72 h time points. Notably, nearly 30−40% of thiopyranone
dioxides 41 and 227 were detected after 72 h compared to less
than 10% for curcumin. Other scaffolds were not investigated
but based on an earlier report;34 the nature of the connecting
linker and substitution on the terminal aryl rings strongly
influenced hydrolytic stability. Thus, the modest stabilities of
thiopyranone dioxides could conceivably be improved by
appropriate modifications at the terminal aryl rings.
3. DISCUSSION
There is growing interest in the pharmacological targeting of
ER stress signaling pathways in cancer.11,17 The ideal agent
should paradoxically aggravate pre-existing stress levels in
tumor cells to the point that prosurvival mechanisms are
overwhelmed and overtaken by the prodeath arm of the UPR.
Curcumin, by inhibiting the degradation of misfolded N-CoR,
The susceptibility of cross-conjugated β-diketone linker in
curcumin to hydrolysis and metabolism has made it a focal
point for structural modification aimed at rectifying these
shortcomings in curcumin. A review of the literature shows that
this moiety is either replaced or altered to give, among others,
Knoevanagel conjugates, monocarbonyl cross-conjugated dien-
ones, and cyclized monocarbonyl cross-conjugated dienones.25
In an earlier investigation, we focused on the APL growth
inhibitory activities of several monocarbonyl cross-conjugated
dienones (monocarbonyls, cyclopentanones, cyclohexanones,
thiopyranones, piperidinones).27 Briefly, we found superior
(submicromolar) activity among the thiopyranones and
piperidinones. These were versatile scaffolds that supported
fluorinated and oxygenated (OH, OCH3) substituents at the
terminal phenyl rings with limited variations in activity. In
contrast, there was a noticeable preference for dioxygenated
(di-OCH3, OH-OCH3) over fluorinated substituents among
monocarbonyls, cyclopentanones, and cyclohexanones. How-
ever, activities were capped at low micromolar levels even for
the most promising analogues. In this report, we explored the
potential of the thiopyranone scaffold by undertaking asym-
metrical substitution of phenyl rings, phenyl to pyridine
replacement, and thiopyranone to thiopyranone dioxide
conversion. The focus on thiopyranone rather than the
equipotent piperidinone scaffold was largely prompted by the
considerable work reported on the latter and which has resulted
in the piperidinone EF24 (42 in this report), arguably one of
the most widely investigated and promising curcumin analogue
identified to date.29,30,42 Of the modifications carried out,
oxidation to give thiopyranone dioxides was the most
promising, yielding compounds (40, 41, 221, 224, 227) with
nanomolar potencies on the APL cell line NB4. In comparison,
the phenyl to pyridine replacement showed only incremental
improvements in activity when compared to the original
thiopyranone leads 32, 34, and 212. Asymmetric substitution of
the terminal phenyl rings of thiopyranone proved disappointing
in that growth inhibitory activity was either no better or even
weaker than the original leads. Several instances of scaffold-
dependent differences in the structure−activity relationship
were noted. For example, thiopyranones showed a regioiso-
meric preference for 2- and 3-pyridinyl terminal rings that
was noticeably absent among previously synthesized cyclo-
hexanones bearing the same phenyl to pyridine replacement. A
comparison of growth inhibitory IC50 NB4 values showed that
the thiopyranone dioxide analogue consistently outperformed
its cyclohexanone, thiopyranone, and piperidinone counter-
parts. Notably, thiopyranone dioxide 40 (IC50 NB4 = 80 nM)
was at least 3 times more potent than piperidinone 42/EF24
(IC50 = 0.29 μM). Taken together, the impressive activities of
the thiopyranone dioxides were clear advantages not observed
with the earlier compounds27 or other scaffolds investigated in
this report.
sensitized APL cells to ER stress-induced apoptosis.16
A
monocarbonyl dienone analogue of curcumin [1E,4E-1,5-
bis(2,3-dimethoxyphenyl)penta-1,4-dien-3-one] acted in a
mechanistically related manner on human lung cancer H460
cells.43 Thus, we rationalized that the potent growth inhibitory
activities of the thiopyranone dioxides on NB4 cells could
likewise be linked to the induction of ER-stress induced
apoptosis. To that end, we have shown that the thiopyranone
dioxide 41 induced build-up of misfolded N-CoR in the cytosol
of NB4 cells. Elevated levels of chaperone proteins (PDI,
GRP73, HSP60) and ER stress sensors (PERK, IRE1, and
constituent proteins) implicated heightened levels of ER stress
in NB4 cells treated with 41. Notably, the proteins in the PERK
and IRE1 signaling arms of the UPR remained elevated for up
to 48 h, pointing to sustained stress levels in the cells.
Furthermore, increases in GADD34 and ASK1, which are critical
to the commitment phase of ER stress-induced apoptosis, signaled
a transition of the UPR from a prosurvival to a prodeath mode.
Microarray analysis of over 28 000 genes in NB4 cells confirmed
that 41 preferentially up-regulated protein processing genes that
were relevant to the activation of the UPR. Taken together, there
is corroboratory evidence pointing to a consequential role for the
UPR in the induction of apoptosis by 41 in NB4 cells.
However, the question remains as to the incipient events that
could have triggered the accumulation of misfolded proteins
and the cascade of prodeath reactions described earlier. We
have shown that 41, 219, and 227 were Michael acceptors.
Their reactivities could conceivably interfere with the normal
course of protein folding and hence exacerbate ER stress.
Another possibility is the inhibition of the proteasome which by
curtailing the degradation of misfolded proteins would
compound ER stress with expected consequences. We showed
that 41 inhibited purified 26S proteasome at low micromolar
concentrations and molecular docking indicated that it was well
placed for productive H bonding and π−π and hydrophobic
interactions to the β5 chymotrypsin binding pocket. However,
the discrepancy between the cell based and in vitro proteasomal
inhibitory potencies of 41 indicated the need for more robust
evidence to support proteasomal involvement in intercepting
the degradation of misfolded proteins. Notwithstanding these
mechanistic aspects, the thiopyranone dioxide scaffold is
distinguished from other monocarbonyl dienone analogues of
curcumin by the potent growth inhibitory activities of several
members on malignant APL cells.
4. EXPERIMENTAL SECTION
When evaluated for time-dependent hydrolytic degradation,
the thiopyranone dioxides 41 and 227 were comparable to
thiopyranone 219 but more stable than curcumin at 48 and
4.1. General Details for Chemical Syntheses. Reagents
(synthetic grade or better) were obtained from commercial suppliers
(Sigma-Aldrich Chemical Co. Inc., Singapore; Alfa Aesar, MA, USA)
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1
and used without further purification. H and 13C NMR spectra were
The organic layer was removed in vacuo, and the residue was purified
by column chromatography with hexane/ethyl acetate as eluting
solvents.
recorded on a Bruker ACF (DPX 400 MHz). Chemical shifts are
reported in parts per million (ppm). The residual solvent peak was
used as an internal reference. Analytical thin layer chromatography
(TLC) was performed with Merck precoated TLC plates, silica gel
60F-254, layer thickness of 0.25 mm. Flash chromatography
separations were performed on Merck 60 (0.040−0.063 mm) mesh
silica gel. Nominal mass spectra were analyzed on LcQ Finnigan MAT
mass spectrometer with chemical ionization (APCI) as probe. High
resolution accurate mass spectra were analyzed on Bruker micrOTOF-
QII mass spectrometer with chemical ionization (APCI) as probe.
Spectroscopic data and reaction yield of final compounds are given in
Supporting Information. Purity of final compounds was verified by
combustion analysis (PerkinElmer PRE-2400 elemental analyzer) or
by reverse phase HPLC on two different solvent systems (isocratic
mode). Details are given in Supporting Information. Unless otherwise
stated, combustion analyses for C and H fell within 0.4% of theoretical
values, and compounds analyzed on HPLC were found to be of ≥95%
purity on both solvent systems.
4.2. General Procedure for Syntheses of 201, 206, 207−214,
228, 229 by Aldol Condensation under Basic Conditions. A
solution of the reacting ketone (cyclohexanone, tetrahydrothiopyran-
4-one or piperidin-4-one) (1 mmol) in 0.3% (w/v) NaOH in aqueous
ethanol (50% v/v, 6 mL) was stirred at room temperature (25 °C) in a
round-bottom flask sealed with a rubber septum. A solution of the
substituted benzaldehyde (2.1 mmol) in ethanol (1 mL) was added
dropwise to the stirred solution over 5 min, after which stirring was
continued overnight at room temperature. The precipitated solids
were removed by vacuum filtration, washed with cold ethanol, and
dried under vacuum. In most cases, the product was purified by
column chromatography with hexane/ethyl acetate as eluting solvents.
For 228 and 229, the free base was dissolved in ethyl acetate and
acidified with 6 M HCl to afford the corresponding HCl salt which was
removed by filtration under reduced pressure and washed with ethyl
acetate.
4.3. General Procedure for Syntheses of 215, 216, 230, 231
by Aldol Condensation under Acidic Conditions. A solution of
the reacting ketone (tetrahydrothiopyran-4-one or piperidin-4-one)
(0.5 mmol) in denatured ethanol (2 mL) was stirred at room
temperature (25 °C) in a round-bottom flask sealed with a rubber
septum. Then 6 M HCl (25 μL) was added followed by the
substituted benzaldehyde (1 mmol). The mixture was heated to 30−
60 °C on an oil bath, and the progress of the reaction was periodically
monitored by TLC over 18−24 h. Solids were removed by vacuum
filtration and washed with ethanol. In most cases, the product was
purified by column chromatography with hexane/ethyl acetate as
eluting solvents followed by recrystallization in suitable solvents.
4.4. General Procedure for Syntheses of 40, 41, 221−227.
The corresponding 3,5-bis(substituted benzylidene)-
tetrahydrothiopyran-4-one required for the syntheses of 40, 41, 221,
222, and 226 were synthesized as reported.23 The 3,5-bis(substituted
benzylidene)tetrahydrothiopyran-4-ones 211, 213−215 were the
starting materials for 223−225, 227. In a dry round-bottom flask
was added the 3,5-bis(substituted benzylidene)tetrahydrothiopyran-4-
one (0.5 mmol) in glacial acetic acid (3.5 mL) which was then stirred
at 30−40 °C, followed by addition of 30% hydrogen peroxide
(320 μL), The mixture was stirred at 60 °C for 6−24 h during which
time the reaction mixture was periodically monitored by TLC. The
flask was cooled to room temperature, distilled water was added, and
the mixture was cooled further in ice to induce the precipitation of the
product as a yellow solid. It was removed by filtration, washed with
distilled water, purified by column chromatography with hexane/ethyl
acetate as eluting solvents, and recrystallized in suitable solvents.
4.5. 2-(3-Fluorobenzylidene)cyclohexanone (1). A solution of
cyclohexanone (4 mmol) in ethanol (4 mL) was stirred at 65 °C.
3-Fluorobenzaldehyde (1 mmol) in ethanol (1 mL) was added dropwise
to the stirred solution over 5 min, followed by 6 M HCl (10 μL).
Stirring was stopped when the starting benzaldehyde was not detected
by TLC. The mixture was evaporated under reduced pressure and the
residue was dissolved in dichloromethane and extracted with brine.
4.6. (3-[(3-Fluorobenzylidene)dihydro-2H-thiopyran-4(3H)-
one) (2). To a clear and dry three-necked round-bottom flask was
added lithium diisopropylamide (LDA) in tetrahydrofuran (THF)
(2 M, 4 mmol). The mixture was cooled to −78 °C in a dry ice and
acetone slurry, and the flask was purged with argon for 3−5 min.
Tetrahydrothiopyran-4-one (2.0 mmol) was dissolved in anhydrous
THF (1 mL) and added dropwise to the stirred LDA mixture at
−78 °C over an argon atmosphere. After 1 h, 3-fluorobenzaldehyde
(2 mmol) in anhydrous THF (1 mL) was added dropwise to the
mixture over 10 min. The progress of the reaction was periodically
monitored by TLC over 6 h. THF was removed in vacuo followed by
extraction with brine/dichloromethane to give a yellow oil which was
dissolved in toluene (3 mL). p-Toluenesulfonic acid (0.1 mmol) was
added, and the mixture was stirred at 90 °C over 1.5 h. The mixture was
evaporated under reduced pressure and worked up with dichloro-
methane and brine to give the crude product which was purified by
column chromatography with hexane/ethyl acetate as eluting solvents.
4.7. General Procedure for Syntheses of 202−205, 217−220.
The procedure described in section 4.3 was followed with minor
changes. A solution of the ketone 1 or 2 (0.5 mmol) in denatured
ethanol (2 mL) was stirred at room temperature (25 °C) in a round-
bottom flask sealed with a rubber septum. Then 6 M HCl (25 μL) was
added followed by the substituted benzaldehyde (0.5 mmol). The
mixture was heated to 65 °C on an oil bath, and the progress of the
reaction was periodically monitored by TLC over 1−2 days. The
mixture was evaporated under reduced pressure and worked up with
dichloromethane and brine to give the crude product which was
purified by column chromatography with hexane/ethyl acetate as
eluting solvents.
4.8. Hydrolytic Stability. The method described by Liang et al.34
was followed with some modifications. Test compound (1 mg) was
dissolved in 0.5 mL of PBS (1×, pH 7.4) comprising DMSO (50%) and
sodium carboxymethylcellulose (0.3%) and kept in the dark at 25 °C.
Aliquots were withdrawn at the stated time period (0, 24, 48, 75 h),
diluted 100× with methanol, and analyzed by HPLC on a Shimadzu
SPD-20A HPLC system. Samples were separated on a Poroshell 120
EC-C18 column (Agilent Technologies) at 80% methanol, 20% H2O;
flow rate of 0.6 mL/min. The % degradation was assessed from a
comparison of peak areas of signals attributed to test compound at the
start of experiment and after x = 24, 48, and 75 h.
(peak area at 0 h) − (peak area at x h)
% degradation =
× 100
peak area at 0 h
The determinations were made on two freshly prepared solutions of test
compound. Results were analyzed for statistical significance using one-way
ANOVA with post-Bonferroni multiple comparison test (GraphPad
Prism, version 3.0).
4.9. NMR Spectroscopic Assay. The method described by
Avonto et al.39 was followed with some modifications. Test compound
(4 mg) was dissolved in 0.5 mL of DMSO-d6, and the 1H NMR
spectrum was recorded immediately on a 400 MHz Bruker ACF NMR
instrument. Cysteamine (6 mg) was added, and the mixture was
1
vortexed for 5 min after which the H NMR spectrum was recorded.
The residual solvent peak was used as an internal reference. NMR
spectra were analyzed using MestRec Research Software (Bajo, Spain).
4.10. Cell Lines and Reagents. The all-trans-retinoic acid
sensitive acute promyelocytic leukemia cell line NB4 and its resistant
variant NB4-R1 were generous gifts from Dr. Y. Homma (Department
of Biosignal Research, Tokyo Metropolitan Institute of Gerontology,
Japan) and Dr. M. Lanotte (INSERM U-301, Centre G. Hayem,
̂
Hopotal Saint-Louis, France), respectively. Leukemic cell lines were
maintained in Rosewell Park Memorial Institute 1640 (RPMI, Gibco)
medium, IMR90 in Eagle’s minimum essential medium (EMEM,
Sigma-Aldrich), and MCF10A in Dulbecco’s modified Eagle medium
(DMEM, Gibco) containing penicillin/streptomycin (50 U/10 μg/mL)
and heat-inactivated fetal bovine serum (10%) at 37 °C in a humidified
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5% CO2 atmosphere. They were used within 10 passages for biological
assays. Buffers and reagents were obtained from Invitrogen Life
Technologies (CA, USA) unless otherwise stated. Curcumin was
purchased from Sigma-Aldrich (Singapore) and dissolved in DMSO.
CellTiter 96 AQueous One solution assay was obtained from Promega
(Madison WI). 7-Amino-4-methylcoumarin (AMC) conjugated fluoro-
genic peptide substrates Boc-Leu-Arg-Arg-AMC, Suc-Leu-Leu-Val-Tyr-
AMC, Z-Leu-Leu-Glu-AMC (for the proteasomal trypsin-like, chymo-
trypsin-like, and peptidylglutamyl peptide hydrolyzing (PGPH or
caspase)-like activities, respectively) were purchased from Biomol,
Enzo Life Sciences Inc. (Farmingdale, NY). The in vitro activity of
human 26S proteasome (Biomol, Enzo Life Sciences, Farmingdale, NY)
was evaluated on the Proteasome Glo Assay System (Promega,
Madison, WI).
Primary antibodies anti-ASK1 (rabbit, 1:1000), anti-caspase 3
(rabbit, 1:1000), anticleaved caspase 3 (rabbit, 1:1000), anti-eIF2α
(rabbit, 1:1000), anti-PARP (rabbit, 1:1000), anti-phospho ASK1
(rabbit, 1:2000), anti-phospho eIF2α (rabbit, 1:1000), anti-phospho
SAPK/JNK (mouse, 1:1000), anti-SAPK/JNK (mouse, 1:1000) were
purchased from Cell Signaling. Antibody anti-β-actin (mouse,
1:10000) was purchased from Sigma. Antibodies anti-N-CoR C-20
(goat, 1:500), anti-GADD34 (rabbit, 1:1000), anti-GRP78 (goat,
1:2000), anti-HSP60 (rabbit, 1:5000), anti-PERK (rabbit, 1:2000),
anti-phosphoPERK (rabbit, 1:2000) were purchased from Santa Cruz
Biotechnology. Secondary horseradish peroxidase antibodies including
goat anti-mouse, goat anti-rabbit, mouse-antigoat (1:10 000) were
purchased from Zymed Laboratories.
4.11. Cell Viability Assay. Effect of test compounds on viability of
cells (NB4, NB4-R1) were determined by the MTS [3-(4,5-
dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-
2H-tetrazolium] assay. Leukemic cells were seeded at 3500 cells per
well, respectively. Stock solutions of test compounds were prepared in
DMSO and diluted with respective media to give a final concentration
of 0.5% v/v DMSO. Cells were treated with at least six concentrations
of test compound for 72 h at 37 °C in a humidified 5% CO2
atmosphere. Concentrations investigated span the dynamic range of
the inverted S-shaped cell viability curve. After this time, 10 μL of
MTS (CellTiter 96 AQueous One solution assay) was added to each
well under dim light. The plates were incubated in the dark at 37 °C
for 4 h, after which absorbance readings were taken at 490 nm on a
microplate reader (Biorad Ultramark microplate imaging system). Cell
viability was determined from the expression
calibration and compensation. 20 000 cells were read for each sample
determination. Each compound was evaluated in at least three
independent experiments using two or more freshly prepared stock
solutions of test compound.
4.13. Western Blotting. The detection of N-CoR by Western
blotting was carried out as described in an earlier report.27 For the
other proteins, the following method was followed. Treated NB4 cells
were harvested as described in section 4.11 except that cell lysis buffer
(Sigma-Aldrich C2978) containing cOmplete protease inhibitor (1×,
Roche) was added. Crude lysates (1 μg/mL) were stored at −80 °C.
Protein samples were separated on SDS−PAGE using 8%, 10%, or
12% resolving gel and gradient voltage of 80 V for the first 30 min
followed by a gradual increase to 100 V over 30 min and then fixed at
100 V for the remaining time. Separated protein samples were
transferred onto prewetted PVDF membrane using a wet trans-
electroblotting system (Bio-Rad Inc., England) at a constant current of
80 V (120 min, 4 °C) using transfer buffer. Methanol (10% v/v) was
added to the transfer buffer prior to use. The membrane was blocked
with 5% milk or BSA in PBS or TBS containing 0.1% v/v Tween
20 (PBS-T or TBS-T) for 1 h, 25 °C followed by incubation
(overnight, 4 °C) with the respective primary antibodies in 5% milk or
BSA in PBS-T or TBS-T with slow shaking. The membrane was
washed with PBS-T or TBS-T (3×, 10 min per wash) and incubated
with horseradish peroxidase-conjugated secondary antibody in 5% milk
or BSA in PBS-T or TBS-T (1 h, 25 °C). The unbound secondary anti-
bodies were removed by washing with PBS-T or TBS-T (3×, 10 min per
wash). The immunoreactive bands were detected by Western Lightning
Chemiluminescence Reagent Plus (PerkinElmer) using a Konica Minolta
SRX-101A film processor.
4.14. Immunostaining and Fluorescence Microscopy for
Detection of N-CoR in Nuclear and Cytosolic Compartments in
NB4 Cells. A previously reported method was followed.27
4.15. Microarray Chip Hybridization and Pathway Analysis.
Total RNA was isolated from treated cells using the RNeasy Plus Mini
kit (Qiagen GmBH, Hilden, Germany) following the manufacturer’s
protocol. mRNA concentrations were measured using the Nanodrop
1000 (Thermo Scientific), and samples with OD260nm/OD280nm
between 1.9 and 2.1 were subjected to electrophoresis on a 1%
agarose gel. The gel was stained with ethidium bromide to determine
the presence of 25S and 16S rRNA. Samples were submitted to
Affymetrix Origin Laboratories (Singapore) for a total RNA quality
check using the Agilent bioanalyzer, after which the samples were
hybridized onto Affymetrix human gene 1.0 ST arrays. Data processing
was carried out by Sciencewerke Pte Ltd. (Singapore) and involved
calibrating the background level and normalizing/transforming the
intensities of the probes. Briefly, the bioconductor package Limma was
used to assess differential expression between control and treated
samples. The Benjamini & Hochberg multiple testing adjustment
method was applied to the p-values, and genes with adjusted p-values
of <0.05 were used for further gene expression comparison between 41
and curcumin. The KEGG pathway database was used to analyze the
profiles of these genes
4.16. Validation by Real-Time PCR. RNase-free water was added
to 2 μg of RNA and 3 pmol of oligo-dT (18-mer) to give a final
volume of 21 μL. The mixture was incubated at 65 °C, 15 min and
immediately quenched on ice thereafter. A mastermix of 10 μL of 5×
RT buffer, 1 μL of 25 mM dNTPs, 0.5 μL of RNasin inhibitor, 1 μL of
murine reverse transcriptase and sterile water was prepared to a total
volume of 29 μL for each sample. The mastermix was added to the
RNA and mixed. The sample was incubated at 42 °C, 1 h for the
generation of cDNA. PCR amplification was carried out in 50 μL
volumes containing 1 μL of 10× template, 1× PCR reaction buffer,
200 nM each dNTP, 0.8 μM each primer, and 1.5 units of DNA
polymerase. Real-time PCR of selected genes was carried out using the
TaqMan gene expression assay system (Applied Biosystems, CA, USA)
and recorded using the ABI 7500 fast PCR system (Applied Biosystems,
CA, USA). The qRT -PCR reaction conditions and list of TaqMan
primers are given in Supporting Information, Tables S2 and S3.
4.17. In Vitro Activity of Human 26S Proteasome. The activity
of human 26S proteasome was evaluated using proteasome Glo
A − C
U − Z
cell viability (%) =
× 100
where A is the absorbance of wells containing cells and test compound,
C is the absorbance of test compound at 490 nm, U is the absorbance
of untreated cells in medium and 0.5% DMSO, and Z is the
absorbance of medium and 0.5% DMSO. The IC50 of test compound
(concentration required to reduce cell viability to 50% of control
values from untreated cells) was determined using GraphPad Prism
(version 3.0, GraphPad software, San Diego, CA). Each concentration
of test compound was evaluated in triplicate for each run, and the assay
was carried out on at least three separate occasions using at least two
freshly prepared stock solutions.
4.12. Apoptosis Determination Using Flow Cytometry. The
annexin V-FITC apoptosis detection kit (Sigma-Aldrich, APOAF) was
used. NB4 cells were seeded at 105 cells/mL and treated with
compounds at respective concentrations (final 0.1% DMSO). After
incubation at 37 °C in a humidified 5% CO2 atmosphere for 48 h,
the cells were transferred to a 50 mL Falcon tube, pelleted by
centrifugation (200g, 5 min), and washed twice with ice-cold 1× PBS.
The cell pellet was declumped thoroughly and resuspended in
1× binding buffer at density of 106 cells/mL. Annexin V-FITC
conjugate protein (5 μL) and propidium iodide solution (1 μL) were
added to cell suspension (500 μL) and incubated at room temperature
for 10 min in the dark. Flow cytometric analysis was carried out on the
Cytomation Cyan LX instrument (Dako, Fort Collins, Co, USA) using
the Summit software. Unstained treated cells and treated cells single-
stained with annexin V-FITC or propidium iodide only were used for
L
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3-substrate system following the manufacturer’s instructions. The kit
comprised luminogenic substrates for chymotrypsin-like (Suc-LLVY-
aminoluciferin), trypsin-like (Z-LRR-aminoluciferin), and caspase-like
(Z-nLPnLD-aminoluciferin) activities. Substrate cleavage liberates
aminoluciferin, which in the presence of luciferase is oxidized to
oxyluciferin with emission of light. The enzyme (1 μg/mL) was
incubated with test compound (41, curcumin, or bortezomib) for 2 or
4 h at 25 °C, after which the substrate-containing proteasomal Glo
reagent was added and luminescence measured after 30 min. Readings
in the presence of test compound were corrected for background
luminescence (blank) and expressed as a % of control luminescence
(absence of test compound) for the determination of IC50.
Determinations were repeated thrice.
4.18. Inhibition of 26S Proteasome Activity in NB4 Cells. The
proteasomal inhibition assay described earlier27 was followed with
some modifications. NB4 cells were seeded at 105 cells/mL and
treated with a known concentration of test compound (final
concentration of DMSO, 0.1%). After incubation at 37 °C in a
humidified 5% CO2 atmosphere for 48 h, the cells were transferred to
a 50 mL Falcon tube, pelleted by centrifuging (200g, 5 min), and
washed twice with ice-cold 1× PBS. The cells were then transferred to
a microcentrifuge tube and 4 times the cell pellet volume of cell lysis
buffer (Sigma-Aldrich C2978) was added. The cells were subjected to
gentle homogenization using a 27G needle and syringe and allowed to
stand over ice with frequent agitation for 30 min. The lysates were
centrifuged at 10 000 rpm, 5 min, 4 °C. Protein concentration in the
supernatant was determined and normalized using BCA protein assay kit
(Pierce Laboratories, IL, USA). Proteasomal activity was determined
immediately using 2 μg (10 μL) of total protein per well. Substrates that
were specific for each catalytic site of the proteasome were Boc-Leu-Arg-
Arg-AMC (60 μM) for trypsin-like, Suc-Leu-Leu-Val-Tyr-AMC (50 μM)
for chymotrypsin-like, and Z-Leu-Leu-Glu-AMC (30 μM) for caspase-like
activity. Concentrations of peptide substrates were chosen based on the
Km (Supporting Information, Figure S6) of the catalytic unit. Km
measurement was carried out by measuring the rate of reaction at each
substrate concentration (4−200 μM) with 2 μg (10 μL) of protein lysate.
Km and Vmax of each substrate were calculated with the Michaelis−
Menten equation. Reactions were initiated by substrate addition (90 μL).
The release of the fluorescent tag AMC on hydrolysis of substrate was
monitored for 1 h at λex = 360 nm and λem = 460 nm (25 °C) on an
Infinite 200 microplate reader (Tecan). Representative plots are shown in
Figure S7 (Supporting Information). Inhibition of proteasomal activity
was determined from the reaction velocities (gradients) of treated and
untreated (control) cell lysates.
hydrolytic stabilities of curcumin, 41, 219, and 227 in
phosphate buffer; induction of apoptosis in NB4 cells by 41,
219, and 227; compounds 41, 219, and 227 induced ER stress
and activated UPR-induced apoptosis in NB4 cells; reaction of
curcumin, 41, and 227 with cysteamine; dose-dependent
inhibition of the trypsin-like, caspase-like, and chymotrypsin-
like activities of the proteasome by curcumin and 41; deter-
mination of Km and Vmax of substrates used for determination of
catalytic activities of NB4 cells; inhibition of trypsin-like,
chymotrypsin-like, and caspase-like activities in NB4 cells by
curcumin and 41 at a fixed concentration of 1 μM; biological
pathways in NB4 cells significantly affected by 41 (50 nM) and
curcumin (5 μM), based on KEGG pathway database analysis; list
of genes in the ER protein processing pathway that were
significantly up-regulated by 41 and curcumin; list of genes that
were significantly down-regulated by both 41 and curcumin; real-
time PCR reaction reagents and conditions; list of TaqMan
primers for real-time PCR. This material is available free of charge
via the Internet at http://pubs.acs.org.
AUTHOR INFORMATION
Corresponding Author
*E-mail: phagoml@nus.edu.sg. Phone: 65-65162654.
■
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
This work was supported by grants from the National Medical
Research Council (NMRC) of Singapore to M.-L.G. and M.K.
(Grant EDG10May045), National Institutes of Health Grant
P01 CA116676 (H.F.), and an Emory Winship Cancer Institute
Kennedy seed grant (to Y.D). H.F. is Georgia Research Alliance
Distinguished Investigator. K.-L.T. was supported by a graduate
research scholarship from the Ministry of Education, Singapore,
and the National University of Singapore.
ABBREVIATIONS USED
■
ASK1, apoptosis signal regulating kinase 1; ATF, activating
transcription factor 6; APL, acute promyelocytic leukemia;
elF2α, eukaryotic initiation factor 2; ER, endothelium
reticulum; ERAD, endothelium reticulum associated degrada-
tion; FACS, fluorescence-activated cell sorting; GADD34,
growth arrest and DNA damage inducible protein 34;
GRP78, glucose-regulated protein 78; HSP60, heat shock
protein 60; IκBα, inhibitor of κBα; IKK, inhibitor of κB kinase;
IRE1, inositol-requiring enzyme 1; JNK, c-Jun N-terminal
kinase; KEGG, Kyoto Encyclopedia of Genes and Genomes;
N-CoR, nuclear co-repressor; NF-κB, nuclear factor κB;
OSGEP, O-sialoglycoprotein endopeptidase; PARP, poly
(ADP-ribose) polymerase; PCR, polymerase chain reaction;
PDI, protein disulfide isomerase; PERK, protein kinase
RNA-like endothelium reticulum kinase; PML, promyelocytic
leukemia; RARα, retinoic acid receptor α; TNFα, tumor
necrotic factor α; UPR, unfolded protein response
gradientcontrol − gradienttreated
% inhibition =
× 100
gradientcontrol
The assay was carried out on at least three separate occasions using two
(or more) freshly prepared stock solutions of test compound.
4.19. Molecular Docking. Human 20S proteasome was re-
trieved from the RCSB Protein Data Bank (PDB 1IRU).41 The β5
chymotrypsin catalytic subunit was identified and processed for
docking using LigX in the software Molecular Operating Environment
(MOE, version 2011, Chemical Computing Group, Montreal,
Canada). Test molecules (41, 206, 211, 219, 220, 221, 227, curcumin)
were also prepared on MOE. Docking simulation was carried out on
GOLD, version 5.2 (Cambridge Crystallographic Data Centre Software
Ltd., Cambridge, U.K.) with default GA settings. The binding pocket was
defined by the atoms within 10 Å radius of the Thr1 hydroxyl oxygen
atom. GOLD uses a genetic algorithm (GA) for docking flexible ligands
into the binding pocket to explore the full range of ligand conformational
flexibility.44 The GOLD score was used to select the best docked
conformations of test compounds in the binding pocket.
REFERENCES
■
(1) Kakizuka, A.; Miller, W. H., Jr.; Umesono, K.; Warrell, R. P., Jr.;
Frankel, S. R.; Murty, V. V.; Dmitrovsky, E.; Evans, R. M.
Chromosomal translocation t(15;17) in human acute promyelocytic
leukemia fuses RAR alpha with a novel putative transcription factor,
PML. Cell 1991, 66, 663−674.
ASSOCIATED CONTENT
■
S
* Supporting Information
Spectroscopic data of synthesized compounds; evaluation of
compound purity by reverse phase HPLC; time dependent
(2) de The, H.; Lavau, C.; Marchio, A.; Chomienne, C.; Degos, L.;
Dejean, A. The PML-RARa fusion mRNA generated by the t(15;17)
M
dx.doi.org/10.1021/jm401352a | J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
translocation in acute promyelocytic leukemia encodes a functionally
altered RAR. Cell 1991, 66, 675−684.
(3) Horlein, A. J.; Naar, A. M.; Heinzel, T.; Torchia, J.; Gloss, B.;
Kurokawa, R.; Ryan, A.; Kamei, Y.; Soderstrom, M.; Glass, C. K.;
Rosenfeld, M. G. Ligand-independent repression by the thyroid
hormone receptor mediated by a nuclear receptor co-repressor. Nature
1995, 377, 397−404.
(4) Heinzel, T.; Lavinsky, R. M.; Mullen, T. M.; Soderstrom, M.;
Laherty, C. D.; Torchia, J.; Yang, W.-M.; Brard, G.; Ngo, S. D.; Davie,
J. R.; Seto, E.; Eisenman, R. N.; Rose, D. W.; Glass, C. K.; Rosenfeld,
M. G. A complex containing N-CoR, mSin3 and histone deacetylase
mediates transcriptional repression. Nature 1997, 387, 43−48.
(5) Laherty, C. D.; Yang, W.-M.; Sun, J. M.; Davie, J. R.; Seto, E.;
Eisenman, R. N. Histone deacetylases associated with the mSin3 Co-
repressor mediate mad transcriptional repression. Cell 1997, 89, 349−
356.
2 by curcumin induces ER stress-associated apoptosis for treating
human liposarcoma. Mol. Cancer Ther. 2011, 10, 461−471.
(21) Wu, S. H.; Hang, L. W.; Yang, J. S.; Chen, H. Y.; Lin, H. Y.;
Chiang, J. H.; Lu, C. C.; Yang, J. L.; Lai, T. Y.; Ko, Y. C.; Chung, J. G.
Curcumin induces apoptosis in human non-small cell lung cancer
NCI-H460 cells through ER stress and caspase cascade- and
mitochondria-dependent pathways. Anticancer Res. 2010, 30, 2125−
2133.
(22) Anand, P.; Kunnumakkara, A. B.; Newman, R. A.; Aggarwal, B.
B. Bioavailability of curcumin: problems and promises. Mol.
Pharmaceuticals 2007, 4, 807−818.
(23) Sharma, R. A. Phase I clinical trial of oral curcumin: biomarkers
of systemic activity and compliance. Clin. Cancer Res. 2004, 10, 6847−
6854.
(24) Yallapu, M. M.; Jaggi, M.; Chauhan, S. C. Curcumin
nanoformulations: a future nanomedicine for cancer. Drug Discovery
Today 2012, 17, 71−80.
(25) Esatbeyoglu, T.; Huebbe, P.; Ernst, I. M.; Chin, D.; Wagner, A.
E.; Rimbach, G. Curcuminfrom molecule to biological function.
Angew. Chem., Int. Ed. 2012, 51, 5308−32.
(26) Agrawal, D. K.; Mishra, P. K. Curcumin and its analogues:
potential anticancer agents. Med. Res. Rev. 2010, 30, 818−860.
(27) Tan, K.-L.; Koh, S.-B.; Ee, R. P.-L.; Khan, M.; Go, M.-L.
Curcumin analogues with potent and selective anti-proliferative
activity on acute promyelocytic leukemia: involvement of accumulated
misfolded nuclear receptor co-repressor (N-CoR) protein as a basis for
selective activity. ChemMedChem. 2012, 7, 1567−1579.
(28) Brown, A.; Shi, Q.; Moore, T. W.; Yoon, Y.; Prussia, A.;
Maddox, C.; Liotta, D. C.; Shim, H.; Snyder, J. P. Monocarbonyl
curcumin analogues: heterocyclic pleiotropic kinase inhibitors that
mediate anticancer properties. J. Med. Chem. 2013, 56, 3456−3466.
(29) Kasinski, A. L.; Du, Y.; Thomas, S. L.; Zhao, J.; Sun, S. Y.; Khuri,
F. R.; Wang, C. Y.; Shoji, M.; Sun, A.; Snyder, J. P.; Liotta, D.; Fu, H.
Inhibition of IkappaB kinase-nuclear factor-kappaB signaling pathway
by 3,5-bis(2-flurobenzylidene)piperidin-4-one (EF24), a novel mono-
ketone analog of curcumin. Mol. Pharmacol. 2008, 74, 654−661.
(30) Thomas, S. L.; Zhao, J.; Li, Z.; Lou, B.; Du, Y.; Purcell, J.;
Snyder, J. P.; Khuri, F. R.; Liotta, D.; Fu, H. Activation of the p38
pathway by a novel monoketone curcumin analog, EF24, suggests a
potential combination strategy. Biochem. Pharmacol. 2010, 80, 1309−
1316.
(31) Olivera, A.; Moore, T. W.; Hu, F.; Brown, A. P.; Sun, A.; Liotta,
D. C.; Snyder, J. P.; Yoon, Y.; Shim, H.; Marcus, A. I.; Miller, A. H.;
Pace, T. W. Inhibition of the NF-kappaB signaling pathway by the
curcumin analog, 3,5-bis(2-pyridinylmethylidene)-4-piperidone
(EF31): anti-inflammatory and anti-cancer properties. Int. Immuno-
pharmacol. 2012, 12, 368−377.
(32) Lanotte, M.; Martin-Thouvein, V.; Najman, S.; Balerini, P.;
Valensi, F.; Berger, R. NB4, a maturation inducible cell line with
t(15;17) marker isolated from a human acute promyelocytic leukemia
(M3). Blood 1991, 77, 1080−1086.
(6) Karagianni, P.; Wong, J. HDAC3: taking the SMRT-N-CoRrect
road to repression. Oncogene 2007, 26, 5439−5449.
(7) Lin, R. J.; Nagy, L.; Inoue, S.; Shao, W.; Miller, W. H., Jr.; Evans,
R. M. Role of the histone deacetylase complex in acute promyelocytic
leukaemia. Nature 1998, 391, 811−814.
(8) Grignani, F.; De Matteis, S.; Nervi, C.; Tomassoni, L.; Gelmetti,
V.; Cioce, M.; Fanelli, M.; Ruthardt, M.; Ferrara, F. F.; Zamir, I.;
Seiser, C.; Lazar, M. A.; Minucci, S.; Pelicci, P. G. Fusion proteins of
the retinoic acid receptor-alpha recruit histone deacetylase in
promyelocytic leukaemia. Nature 1998, 391, 815−818.
(9) Khan, M. M.; Nomura, T.; Chiba, T.; Tanaka, K.; Yoshida, H.;
Mori, K.; Ishii, S. The fusion oncoprotein PML-RAR induces
endoplasmic reticulum (ER)-associated degradation of N-CoR and
ER stress. J. Biol. Chem. 2004, 279, 11814−11824.
(10) Verfaillie, T.; Garg, A. D.; Agostinis, P. Targeting ER stress
induced apoptosis and inflammation in cancer. Cancer Lett. 2010, 332,
249−264.
(11) Schonthal, A. H. Pharmacological targeting of endoplasmic
reticulum stress signaling in cancer. Biochem. Pharmacol. 2013, 85,
653−666.
(12) Ng, A. P. P.; Howe Fong, J.; Sijin Nin, D.; Hirpara, J. L.; Asou,
N.; Chen, C. S.; Pervaiz, S.; Khan, M. Cleavage of misfolded nuclear
receptor corepressor confers resistance to unfolded protein response-
induced apoptosis. Cancer Res. 2006, 66, 9903−9912.
(13) Lane, A. A.; Ley, T. J. Neutrophil elastase cleaves PML-
RARalpha and is important for the development of acute
promyelocytic leukemia in mice. Cell 2003, 115, 305−318.
(14) Lane, A. A.; Ley, T. J. Neutrophil elastase is important for PML-
retinoic acid receptor activities in early myeloid cells. Mol. Cell. Biol.
2004, 25, 23−33.
(15) Szegezdi, E.; Logue, S. E.; Gorman, A. M.; Samali, A. Mediators
of endoplasmic reticulum stress-induced apoptosis. EMBO Rep. 2006,
7, 880−885.
(16) Ng, A. P. P.; Chng, W. J.; Khan, M. Curcumin sensitizes acute
promyelocytic leukemia cells to unfolded protein response-induced
apoptosis by blocking the loss of misfolded N-CoR protein. Mol.
Cancer Res. 2011, 9, 1−11.
(17) Luo, B.; Lee, A. S. The critical roles of endoplasmic reticulum
chaperones and unfolded protein response in tumorigenesis and
anticancer therapies. Oncogene 2013, 32, 805−818.
(33) Nason-Burchenal, K.; Maerz, W.; Albanell, J.; Allopenna, J.;
Martin, P.; Moore, M. A.; Dmitrovsky, E. Common defects of different
retinoic acid resistant promyelocytic leukemia cells are persistent
telomerase activity and nuclear body disorganization. Differentiation
1997, 61, 321−311.
(18) Pae, H. O.; Jeong, S. O.; Jeong, G. S.; Kim, K. M.; Kim, H. S.;
Kim, S. A.; Kim, Y. C.; Kang, S. D.; Kim, B. N.; Chung, H. T.
Curcumin induces pro-apoptotic endoplasmic reticulum stress in
human leukemia HL-60 cells. Biochem. Biophys. Res. Commun. 2007,
353, 1040−1045.
(34) Liang, G.; Shao, L.; Wang, Y.; Zhao, C.; Chu, Y.; Xiao, J.; Zhao,
Y.; Li, X.; Yang, S. Exploration and synthesis of curcumin analogues
with improved structural stability both in vitro and in vivo as cytotoxic
agents. Bioorg. Med. Chem. 2009, 17, 2623−2631.
(35) Hasima, N.; Aggarwal, B. B. Targeting proteasomal pathways by
dietary curcumin for cancer prevention and treatment. Curr. Med.
Chem. 2014, 21, 1583−1594.
(19) Ip, S. W.; Wu, S. Y.; Yu, C. C.; Kuo, C. L.; Yu, C. S.; Yang, J. S.;
Lin, Z. P.; Chiou, S. M.; Chung, H. K.; Ho, H. C.; Chung, J. G.
Induction of apoptotic death by curcumin in human tongue squamous
cell carcinoma SCC-4 cells is mediated through endoplasmic reticulum
stress and mitochondrial-dependent pathways. Cell Biochem. Funct.
2011, 29, 641−650.
(36) MacManus, J. P.; Brewer, L. M.; Whitfield, J. F. The widely-
distributed tumor protein, oncomodulin, is a normal constituent of
human and rodent placentas. Cancer Lett. 1985, 27, 145−151.
(37) Balakrishnan, M. P.; Cilenti, L.; Ambivero, C.; Goto, Y.; Takata,
M.; Turkson, J.; Li, X. S.; Zervos, A. S. THAP5 is a DNA-binding
transcriptional repressor that is regulated in melanoma cells during
(20) Wang, L.; Wang, L.; Song, R.; Shen, Y.; Sun, Y.; Gu, Y.; Shu, Y.;
Xu, Q. Targeting sarcoplasmic/endoplasmic reticulum Ca(2)+-ATPase
N
dx.doi.org/10.1021/jm401352a | J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
DNA damage-induced cell death. Biochem. Biophys. Res. Commun.
2011, 404, 195−200.
(38) Wang, X.; Thomas, B.; Sachdeva, R.; Arterburn, L.; Frye, L.;
Hatcher, P. G.; Cornwell, D. G.; Ma, J. Mechanism of arylating
quinone toxicity involving Michael adduct formation and induction of
endoplasmic reticulum stress. Proc. Natl. Acad. Sci. U.S.A. 2006, 103,
3604−3609.
(39) Avonto, C.; Taglialatela-Scafati, O.; Pollastro, F.; Minassi, A.; Di
Marzo, V.; De Petrocellis, L.; Appendino, G. An NMR spectroscopic
method to identify and classify thiol-trapping agents: revival of Michael
acceptors for drug discovery? Angew. Chem., Int. Ed. 2011, 50, 467−
471.
(40) Milacic, V.; Banerjee, S.; Landis-Piwowar, K. R.; Sarkar, F. H.;
Majumdar, A. P. N.; Dou, Q. P. Curcumin inhibits the proteasomal
activity in human colon cancer cells in vitro and in vivo. Cancer Res.
2008, 68, 7283−7292.
(41) Unno, M.; Mizushima, T.; Morimoto, Y.; Tomisugi, Y.; Tanaka,
K.; Yasuoka, N.; Tsukihara, T. The structure of the mammalian 20S
proteasome at 2.75 Å resolution. Structure 2002, 10, 609−618.
(42) Adams, B.; Ferstl, E.; Davis, M.; Herold, M.; Kurtkaya, S.;
Camalier, R.; Hollingshead, M.; Kaur, G.; Sausville, E.; Rickles, F.
Synthesis and biological evaluation of novel curcumin analogs as anti-
cancer and anti-angiogenesis agents. Bioorg. Med. Chem. 2004, 12,
3871−3883.
(43) Wang, Y.; Xiao, J.; Zhou, H.; Yang, S.; Wu, X.; Jiang, C.; Zhao,
Y.; Liang, D.; Li, X.; Liang, G. A novel monocarbonyl analogue of
curcumin, (1E,4E)-1,5-bis(2,3-dimethoxyphenyl)penta-1,4-dien-3-one,
induced cancer cell H460 apoptosis via activation of endoplasmic
reticulum stress signaling pathway. J. Med. Chem. 2011, 54, 3768−
3778.
(44) Verdonk, M. L.; Cole, J. C.; Hartshorn, M. J.; Murray, C. W.;
Taylor, R. D. Improved protein−ligand docking using GOLD. Proteins
2003, 52, 609−623.
O
dx.doi.org/10.1021/jm401352a | J. Med. Chem. XXXX, XXX, XXX−XXX