The Conjugated Double Bond of Coniferyl Aldehyde Is Essential for Heat Shock Factor 1 Mediated Cytotoprotection

Article abstract of DOI:10.1021/acs.jnatprod.7b00126

Coniferyl aldehyde (1) is previously reported as a potent inducer of heat shock factor 1 (HSF1). Here, we further examined the active pharmacophore of 1 for activation of HSF1 using the derivatives coniferyl alcohol (2), 4-hydroxy-3-methoxyphenylpropanal (3), and 4-hydroxy-3-methoxyphenylpropanol (4). Both 1 and 2 resulted in increased survival days after a lethal radiation (IR) dose. The decrease in bone marrow (BM) cellularity and Ki67-positive BM cells by IR was also significantly restored by 1 or 2 in mice. These results suggested that the vinyl moiety of 1 and 2 is necessary for inducing HSF1, which may be useful for developing small molecules for cytoprotection of normal cells against damage by cytotoxic drugs and radiation.

Full text of DOI:10.1021/acs.jnatprod.7b00126

Note
pubs.acs.org/jnp
The Conjugated Double Bond of Coniferyl Aldehyde Is Essential for
Heat Shock Factor 1 Mediated Cytotoprotection
Seul-Ki Choi,†,# Gil-Im Mun, Eun Choi, Seo-Young Kim, Youngjoo Kwon, Younghwa Na,*
†,#
†
†
†
,‡
,†
and Yun-Sil Lee*
†Graduate School of Pharmaceutical Sciences, Ewha Womans University, Seoul 120-750, Korea
‡College of Pharmacy, CHA University, Pocheon 487-010, Korea
*
S Supporting Information
ABSTRACT: Coniferyl aldehyde (1) is previously reported as a potent inducer of heat
shock factor 1 (HSF1). Here, we further examined the active pharmacophore of 1
for activation of HSF1 using the derivatives coniferyl alcohol (2), 4-hydroxy-3-
methoxyphenylpropanal (3), and 4-hydroxy-3-methoxyphenylpropanol (4). Both 1 and 2
resulted in increased survival days after a lethal radiation (IR) dose. The decrease in bone
marrow (BM) cellularity and Ki67-positive BM cells by IR was also significantly restored
by 1 or 2 in mice. These results suggested that the vinyl moiety of 1 and 2 is necessary for
inducing HSF1, which may be useful for developing small molecules for cytoprotection of
normal cells against damage by cytotoxic drugs and radiation.
eat shock factor 1 (HSF1) is a master regulator of the heat
shock response and enables cell development, survival, and
against radiation or cytotoxic drug-mediated injury during cancer
11
H
therapy or exposure to radiation hazards. Furthermore, after
the Chernobyl disaster (1986) and Fukushima nuclear disaster
(2011) caused radiation damage, such hazards have awakened
proliferation in mammals. HSF1 exists in an inactive condition in
the cytoplasm. After exposure to proteotoxic stresses, heat shock
proteins (HSPs) are expressed by HSF1 and facilitate chaperone
activities and protein refolding. HSF1 trimerizes and translocates
to the nucleus to bind heat shock elements in promoters of target
12
the need to develop radiation protectors. Work on radio-
protective chemicals started a considerable time ago in the
12,13
United States, at the inception of the Manhattan Project.
However, many of the chemicals had severe side effects such as
1
genes and hyperphosphorylation. Expression of HSPs increases
13
with HSF1 activation, which occurs when cells are exposed to
hyperthermia or biological stresses such as heat, high pressure, or
toxic compounds. HSPs protect cells by influencing a variety of
nausea, vomiting, and hypotension. The search for safe and
nontoxic radioprotectors has yielded a great deal of information
14,15
and several clinical trials.
2
cellular processes that determine cell fate. Thus, HSF1 may be a
Many previous phytochemical investigations have resulted in
the isolation of lignans, flavonoids, iridoids, and terpenoids.
Among them, coniferyl aldehyde (1), a phenolic compound
isolated from the bark of Eucommia ulmoides Oliv. (Eucommia-
ceae), showed cytoprotective effects against radiation and
cytotoxic drugs by inducing HSPs, especially HSP27 and
3
−5
pharmacological target for cytoprotection.
Pharmacological regulators of the heat shock response are
small molecules that enhance expression or function of HSPs and
are promising for chronic or acute treatment of human disease.
An important feature of HSPs is cytoprotection and repair of cells
and tissues against deleterious effects of stress and trauma.
Overexpression of one or more HSP genes is sufficient to protect
against lethal exposure to heat, cytotoxic drugs, and tumor
necrosis factor. The protective effect of HSPs is related to
16
HSP70, through activation of HSF1. Therefore, this study
investigated the active sites of 1 for activation of HSF1 using its
derivatives coniferyl alcohol (2), 4-hydroxy-3-methoxyphenyl-
propanal (3), and 4-hydroxy-3-methoxyphenylpropanol (4).
In a previous study, 1, isolated from the bark of E. ulmoides,
increased HSF1 protein and its transcriptional targets hsp25
2
interference with apoptotic pathways.
Radiation therapy is an important modality for treating cancer.
Almost 40% of cancer patients require radiation therapy for
disease management. Although radiation therapy improves the
survival of a significant number of cancer patients, toxicity and
injury of radiotherapy to normal tissues and organs is substantial.
Moreover, recent clinical anticancer therapy has been targeting a
17
(
murine form, hsp27 for human form) and hsp70. Compound 1
time-dependently and dose-dependently induced HSF1 protein,
accompanied by increased HSP27 and HSP70 protein. Com-
pound 1 did not show any discernible cytotoxicity in L132
6,7
decrease in normal cell damage. Several reports have attempted
to develop new mitigators or protectors from nontoxic phyto-
Received: February 21, 2017
8,9
10
chemicals or natural products, to protect normal tissues
©
XXXX American Chemical Society and
American Society of Pharmacognosy
A
DOI: 10.1021/acs.jnatprod.7b00126
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Journal of Natural Products
Note
normal lung cells. However, paclitaxel and cisplatin, which are
effective anticancer drugs, showed significant cytotoxicity effects.
Coniferyl aldehyde (1) regulates HSF1 protein stability, and this
function is mediated by phosphorylation of HSF1. Compound 1
phosphorylates HSF1 on Ser326; however, other serine residues
such as Ser216 and Ser303/307 are not changed by 1. These data
indicated that 1 is responsible for increased protein stability and
it is hypothesized that for the activation of HSF1 the benzene
vinyl moiety of 1 or 2 is essential.
16,17
transcriptional activity by HSF1.
In the present investigation, the active pharmacophore of 1
for activation of HSF1 was examined. For this purpose, 1 and
compounds with similar structures (2−4), which all have
guaiacol (GU, also called 2-methoxyphenol) moieties in their
basic structure, were compared for HSF1 activation. Compounds
3
and 4 were obtained through the general reductive hydro-
genation for 1 using H gas at atmospheric pressure under
2
Indeed, both 1 and 2 increased the promoter activity of hsp25
and hsp70 (Figure 1A). mRNA of hsp27 and hsp70 and their
proteins were also induced by 3 μM 1 and 2 (Figure 1B and C).
When cell viability was measured in an MTT assays, 1 and 2 did
not show any cytotoxicity, even at 20 μM (Figure 1D), while
celastrol showed strong cytotoxicity. Compound 1 was more
cytotoxic than 2 based on normal cell viability. Since 1 is reported
to be phosphorylated at Ser326 of HSF1, it was determined if 2
also phosphorylates HSF1 at Ser326, similar to 1. Both 1 and 2 at
3 μM phosphorylated HSF1 at Ser326, which might be the main
pathway for HSF1 activation (Figure 1E).
10%-Pd/C catalytic conditions in methanol. Stable pGL4-hsp70
or -hsp25 NCI-H460 cells were plated, and after 24 h, luciferase
activity was assayed from cell lysates using luciferase assay kits
and detected by a luminometer. Compounds 1 and 2 at 10 or
30 μM resulted in increased promoter activity, while 3 and 4 did
not (Figure S1A, Supporting Information). Expression of HSPs
such as HSP27 and HSP70 after treatment for 12 h with 10 or
16
30 μM compounds was monitored by Western blots. HSF1 and
HSP proteins were upregulated by 1 and 2. However, with 3 and 4,
which do not have a vinyl moiety, the HSF1, HSP27, and HSP70
proteins did not increase, even at the higher dose of 30 μM
In a previous study, HSF1 and HSPs were upregulated by 1 in a
time- and dose-dependent manner, with maximal induction at
(
Figure S1B, Supporting Information). Celastrol (1 μM), an HSP
16
inducer, was used as the positive control. These data indicated that
only 1 and 2, a hydroxy derivative of 1, induced promoter activity
of hsp25 and hsp70 and the HSF1 and HSP proteins. Therefore,
3 μM and 12 h. Compound 2 also induced an increase in HSF1
and HSPs, similar to 1. HSF1 induction was more prominent
in 1-treated cells. Time-dependent expression of HSP27 was
Figure 1. Compound 2 is a hydroxy derivative of 1 with similar effects to 1 for inducing HSF1 and HSPs. (A) HSP promoter activity after treatment with
and 2. NCI-H460 cells were transfected with luciferase reporter construct with the hsp25 or hsp70 promoter. Values are expressed as the means ± SD
1
of at least three independent experiments in the form of fold-change relative to the negative control. (B) Immunoblotting was performed 12 h after
treatment with 3 μM 1 or 2 in L132 human lung fibroblast cells. (C) RT-PCR with the same conditions as immunoblotting. (D) MTT assays were
performed 24 h after treatment with the indicated doses of 1 and 2. (E) Phosphorylation patterns of HSF1 after 12 h of treatment of 1 or 2 were detected
by immunoblotting. (F) Immunoblotting was performed 12 h after 1 or 2 treatment or with (G) 3 μM 1 and 2 at the indicated times. Band intensities
were measured and normalized to loading control bands. Experiments were performed for at least three independent experiments. Values are presented
as means ± SD. Celastrol (Cela, 1 μM) was used as the positive control.
B
DOI: 10.1021/acs.jnatprod.7b00126
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Note
Figure 2. Cytoprotective effect of 1 and 2 against paclitaxel, cisplatin, and IR damage. Cell death by immunoblotting of apoptosis-related cleavage of
PARP and caspase-3 (A, C) or FACS (B, D) after 24 h treatment with 0.5 μM paclitaxel or 20 μM cisplatin or 48 h of 10 Gy IR in L132 cells (A, B) or
HSF1+/+ and HSF1−/− MEF cells (C, D) with or without pretreatment with 1 and 2 (*p < 0.05; **p < 0.01). Band intensities were measured and
normalized to loading control bands. Values are expressed as means ± SD of at least three independent experiments in the form of fold-change relative to
the negative control.
more prominent in 2-treated cells, while the dose-dependence
of HSP27 expression was more obvious with 1-treated cells.
For HSP70 expression, neither 1 nor 2 had strong effects.
The results indicated that both 1 and 2 increased expression of
HSF1 and HSPs (Figure 1F and G).
mortality within 22 days. A single treatment of 1 or 2 before IR or
three treatments with 1 and 2 after IR at 5 or 10 mg/kg increased
mean survival days and reduced radiation sickness characteristics
compared to mice given IR alone (Figure 3A). Normal mice
treated with 1 or 2 at 10 mg/kg did not exhibit any noticeable
signs of toxicity within 30 days.
To examine if HSF1 induction by 1 and 2 affected cell damage
induced by paclitaxel, cisplatin, or IR, 1 and 2 were used at 3 μM
for pretreatment followed by incubation with or without 0.5 μM
paclitaxel, 20 μM cisplatin, or IR 10 Gy. To determine induction
of apoptosis, cleavage of PARP and caspase-3 was examined.
To determine cell survival, FACS was performed. Treatment of
L132 cells with paclitaxel and cisplatin for 24 h or IR for 48 h
increased cleavage of PARP and caspase-3. One-hour pretreat-
ment with 1 or 2 before paclitaxel, cisplatin, or IR reduced the
cleavage (Figure 2A). Paclitaxel, cisplatin, or IR was cytotoxic to
L132 cells, but a 1 h pretreatment with 3 μM 1 or 2 inhibited
paclitaxel-, cisplatin-, or IR-mediated cytotoxicity. These data
indicated that pretreatment with 1 or 2 protected cells against
damage from toxic drugs or IR (Figure 2B). To determine
whether 1- and 2-mediated cytoprotective effects depended on
HSF1 expression, HSF1−/− and HSF1+/+ cells were tested.
One-hour pretreatment with 1 or 2 reduced paclitaxel, cisplatin,
or IR cytotoxicity and apoptosis in HSF1+/+ cells at 24 h after
paclitaxel and cisplatin treatment and 48 h after IR exposure.
In HSF1−/− cells, 1 and 2 did not inhibit paclitaxel, cisplatin, or
IR cytotoxicity and apoptosis (Figure 2C and D). These results
suggested that 1- and 2-mediated cyto- or radioprotection was
dependent on HSF1 activation.
Bone marrow (BM) damage after a sublethal dose of IR
(
4.5 Gy) was also examined. At 9 days, IR significantly induced
BM damage; however, 1 or 2, at 10 mg/kg, inhibited IR-induced
BM damage (Figure 3B). IR significantly reduced circulating
white blood cells (WBCs), neutrophils, and lymphocytes after
9
days, and recovery of this parameter was observed following
combined treatment with 1 or 2 (Figure 3C). Effects were more
obvious in 2-treated mice than 1-treated mice except for neutro-
phils. Cellular proliferation was also examined using immuno-
histochemistry of Ki67 with BM tissues, and combined treatment
with 1 or 2 reduced IR-mediated inhibition of Ki67-positive cells
in BM (Figure 3D). When the weight of spleens was examined,
reduced spleen weights by IR were mitigated by combined
treatment with 1 or 2 (Figure 3E).
HSF1 activation by treatment with 1 or 2 may be an obstacle
for radio- and chemotherapy of tumors. However, this possibility
can be possibly excluded because a previous study has indicated
that treatment of 1 did not affect IR-induced cell death in lung
16
tumor tissues in an orthotopic lung tumor model. Although 1
and 2 had similar effects, 1 exhibited more cytotoxicity than 2,
based on cell viability, suggesting a greater clinical promise of 2
rather than 1.
16
Compound 1 increases survival after lethal doses of IR.
Thus, it was determined whether the cytoprotective effects by 2
would be conserved in an animal model like 1 by investigating the
survival after exposure to a lethal dose of IR (7 Gy). IR alone
resulted in signs of radiation sickness such as reduced intake of
food and water, irritability, and lethargy at 10 days with complete
In summary, the present study identified an active pharma-
cophore for activating HSF1, which may be useful for developing
small molecules for cytoprotection against damage by drugs such
as paclitaxel and cisplatin and by radiation of normal cells.
C
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Figure 3. Effect of 1 and 2 on survival days after 7 Gy lethal IR or 4.5 Gy IR-induced bone marrow (BM) damage in ICR mice. (A) Mice underwent
whole-body irradiation with 7 Gy. Compound 1 or 2 (5 and 10 mg/kg, once before and three times after IR) was injected intraperitoneally (ip). The
number of days survival was recorded. Each group contained 10 mice. (B) Mice received 4.5 Gy whole-body radiation. Compound 1 or 2 (10 mg/kg,
once before and three times after IR) was injected ip. Nine days after IR, mice were sacrificed. Each group contained five mice. After H&E staining of BM,
quantification of BM cellularity using an image analyzer was performed. The graph indicates the percentage (means ± SD) of hematopoietic cells
compared to unit area (200 μm × 200 μm, original magnification 200×) of BM. WBCs, lymphocytes, and neutrophils in the peripheral blood (C) and
Ki67-positive cells of BM (D) were measured. The number of Ki67-positive cells in five fields per femur was counted at an original magnification of
1000×. (E) Relative spleen weights were calculated, with values of control spleens set to 1.0. Data are presented as means ± SD of five mice (*p < 0.05).
EXPERIMENTAL SECTION
β-actin were from Santa Cruz Biotechnology (diluted 1:1000;
Santa Cruz, CA, USA). Anticleaved PARP and caspase-3 were from
Cell Signaling Technology (diluted 1:1000; Danvers, MA, USA).
Plasmids and Cell Culture. Human embryonic lung L132 cells
were from the American Type Culture Collection (ATCC, Manassas,
VA, USA) and cultured in RPMI medium, supplemented with 10%
fetal bovine serum (FBS) and 1% penicillin−streptomycin (Gibco BRL)
■
1
13
General Experimental Procedures. The H and C NMR spectra
were recorded on a Varian NMR AS 400 MHz instrument. Chemical
shifts (δ) are in parts per million (ppm) relative to tetramethylsilane as
an internal standard, and coupling constants (J values) are in hertz. Mass
spectrometric investigations were performed on an AB SCIX API 4000
equipped with an electrospray ionization (ESI) source. The solvents and
reagents used were of the highest commercial grade available and used as
received.
Test Compounds and Chemicals. Coniferyl aldehyde (1; 98%
purity), coniferyl alcohol (2; 98% purity), paclitaxel (purity ≥99%), and
cisplatin (purity ≥99%) were purchased from Sigma-Aldrich (St. Louis,
MO, USA).
Synthesis of Compounds 3 and 4. A reaction mixture of 1 (0.20 g,
.12 mmol) and 10%-Pd/C (catalytic amount) was stirred under a H2
atmosphere (equipped with a balloon) for 2 h and then filtered. After
washing with methanol, the solvent was removed under reduced
pressure. The residue was purified by silica gel column chromatography
at 37 °C in a humidified 5% CO incubator. HSF1 knockout mouse
2
embryonic fibroblasts (HSF1+/+ and HSF1−/− MEF) cells were
kindly provided by Dr. Ivor Benjamin (University of Utah, Salt Lake
City, UT, USA). Cells were cultured in Dulbecco’s modified Eagle’s
medium (Thermo Scientific, Rockford, IL, USA), supplemented
with heat-inactivated 10% FBS (Thermo Scientific), 0.1 mmol/L non-
essential amino acids, glutamine, HEPES, and antibiotics at 37 °C in a
5
% CO humidified incubator.
2
1
Promoter Assay. Promoter activities of hsp25 or hsp70 were eval-
uated using luciferase reporter constructs that stably expressed hsp25 or
5
hsp70. Human lung cancer NCI-H460 cells were plated at 1 × 10 cells in
4
6
0 mm dishes and 2 × 10 cells in a 96-well plate. After 24 h, cells were
(
(
eluant: ethyl acetate−n-hexane, 1:2 → 1:1) to give compounds 3
0.024 g, 11.9%) and 4 (0.12 g, 70.4%).
-Hydroxy-3-methoxyphenylpropanal (3): colorless oil; H NMR
treated with test chemicals and harvested 12 h later. Luciferase activity was
assayed from cell lysates using a luciferase assay system (Promega, Madison,
WI, USA), and the results were normalized to β-galactosidase expression
(Promega). All results were the mean of three independent experiments.
Irradiation. Cells in 60 mm Petri dishes were exposed to γ-radiation
1
4
(
3
6
(
DMSO-d , 400 MHz) δ 2.65 (t, J = 7.6 Hz, 2H), 2.79 (t, J = 7.6 Hz, 2H),
6
.77 (s, 3H), 6.54 (dd, J = 8.0, 2.0 Hz, 1H), 6.61 (d, J = 2.0 Hz, 1H),
.70 (d, J = 8.0 Hz, 1H), 7.56 (s, 1H), 9.72 (t, J = 1.2 Hz, 1); 13C NMR
137
(7 or 10 Gy as a single dose) generated by a Cs gamma-ray source
DMSO-d , 100 MHz) δ 27.4, 45.1, 55.5, 111.4, 114.9, 120.2, 131.3, 144.3,
(Elan 3000, Atomic Energy of Canada, Mississauga, Canada) at a dose
rate of 3.81 Gy/min. Radiation workers annually received radiation
safety management training provided by the Korea Foundation of
Nuclear Safety (KoFONS).
6
+
+
1
47.0, 201.7; ESIMS m/z 181.09 [M + H] (calcd for C H O , 181.09).
-Hydroxy-3-methoxyphenylpropanol (4): colorless oil; H NMR
10 13 3
1
4
(
CDCl , 400 MHz) δ 1.87 (tt, J = 8.0, 6.4 H, 2H), 2.64 (t, J = 8.0 Hz,
3
5
2
6
1
1
H), 3.68 (t, J = 6.4 Hz, 2H), 3.87 (s, 3H), 7.69 (dd, J = 8.4, 1.6 Hz, 1H),
Immunoblotting. Cells were plated at 2 × 10 mm in cell culture
13
.70 (d, J = 1.6 Hz, 1H), 6.83 (d, J = 8.4 Hz, 1H); C NMR (CDCl3,
00 MHz) δ 31.2, 34.6, 55.5, 60.2, 112.5, 115.2, 120.3, 132.9, 144.3,
dishes and grown to 30% confluence. Equal amounts of protein were
dissolved in lysis buffer, and lysates were centrifuged at 13 000 rpm for
30 min at 4 °C. Protein concentration was determined using Bradford
(Bio-Rad, CA, USA) assays with bovine serum albumin for standard
curves. The optical density of standards and samples was read by a
+
+
3
47.3; ESIMS m/z 183.10 [M + H] (calcd for C H O , 183.10).
10
15
Antibodies. Monoclonal antibodies against HSF1 were from Abcam
diluted 1:1000; Cambridge, MA, USA). Anti-HSP70, HSP27, and
(
D
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Journal of Natural Products
Note
microplate reader at 595 nm. Prepared samples were mixed with sample
buffer and boiled at 100 °C for 5 min, and proteins were separated via
sodium dodecyl sulfate-polyacrylamide gel electrophoresis (8% or 12%).
Separated protein bands were transferred to nitrocellulose membranes.
After blocking with 5% skimmed milk in phosphate-buffered saline
with Tween 20 (PBST), membranes were incubated with antibodies for
AUTHOR INFORMATION
■
Corresponding Authors
Tel: +82-31-8017-9896. Fax: +82-31-8017-9620. E-mail:
yna7315@cha.ac.kr (Y. Na).
Tel: +82-2-3277-3022. Fax: +82-2-3277-3051. E-mail:
yslee0425@ewha.ac.kr (Y.-S. Lee).
ORCID
Yun-Sil Lee: 0000-0002-7538-8850
Author Contributions
S.-K. Choi and G.-I. Mun contributed equally to this work.
*
*
1
8 h at 4 °C, washed and incubated with horseradish peroxidase, and
visualized via enhanced chemiluminescence. Membranes were detected
by ChemiDoc MP Systems (Bio-Rad).
Reverse Transcription-Polymerase Chain Reaction (RT-PCR).
Total RNA was extracted using TRIzol (Invitrogen, Grand Island, NY,
USA), and cDNA was synthesized using ReverTra Ace RT-PCR kits
#
(
Toyobo, Osaka, Japan). Hsf1, hsp70, and hsp27 transcripts were
Notes
measured by RT-PCR (GenDEPOT, Barker, TX, USA) with gapdh as
the internal control gene. Change in relative gene expression was
normalized to gapdh mRNA using the NIH ImageJ program.
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
MTT Assay. 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium
bromide (MTT; Sigma-Aldrich) assays were used as an indirect measure
of death. Medium was removed and replaced with 100 μL of MTT
reagent (5 mg/mL) in PBS. Plates were incubated for 4 h at 37 °C, and
This work was supported by grants (2015M2A2A7A 03044831
and 2017R1A2B2002327) of the National Research Foundation
of Korea (NRF), funded by the Korean government (Ministry of
Science, ICT & Future Planning).
1
00 μL of DMSO was added to dissolve formazan crystals. Multiwell
plates were shaken for 15 min, and signals detected using ELISA at
40 nm. MTT assay results were expressed as cell numbers by
5
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0 min. Nonspecific binding was prevented by incubation with 5% normal
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human Ki67 antibody (1:100 dilutions) as the primary antibody overnight
at 4 °C. Secondary antibody incubation and staining were performed using
a Vectastain ABC kit (Vector Laboratories, Burlingame, CA, USA).
Statistical Analysis. All results represent means ± SD of three
independent experiments. Statistical significance of differences between
untreated controls and treated groups was compared by Student’s t test.
ASSOCIATED CONTENT
Supporting Information
The Supporting Information is available free of charge on the ACS
Publications website at DOI: 10.1021/acs.jnatprod.7b00126.
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Additional information (PDF)
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DOI: 10.1021/acs.jnatprod.7b00126
J. Nat. Prod. XXXX, XXX, XXX−XXX