ORIGINAL ARTICLE – TRANSLATIONAL RESEARCH AND BIOMARKERS
Resveratrol Induces Notch2-Mediated Apoptosis and Suppression
of Neuroendocrine Markers in Medullary Thyroid Cancer
Matthew Truong, BA1,2, Mackenzie R. Cook, MD1,2, Scott N. Pinchot, MD1,2, Muthusamy Kunnimalaiyaan, PhD1,2,
and Herbert Chen, MD, FACS1,2,3
1Department of Surgery, Endocrine Research Laboratory, University of Wisconsin, Madison, WI;2University of
Wisconsin Carbone Cancer Center, Madison, WI;3H4/722 Clinical Science Center, University of Wisconsin, Madison, WI
Background. Currently, complete surgical resection is the
only curative option for medullary thyroid cancer (MTC).
Previous work has shown the Notch pathway is a potent
tumor suppressor in MTC and that resveratrol activates the
Notch pathway in carcinoid cancer, a related neuroedocrine
malignancy. In this study, we hypothesized that the effects
observed on carcinoid cells could be extended to MTC.
Methods. MTC cells treated with varying doses of res-
veratrol were assayed for viability by the MTT (3-[4,5-
dimethylthiazol-2-yl]-2,5 diphenyl tetrazolium bromide)
assay. Western blot analysis for achaete-scute complex-like
1 (ASCL1), chromogranin A (CgA), full-length and
cleaved caspase 3, and poly-ADP ribose polymerase
(PARP) was performed. Quantitative real-time polymerase
chain reaction (qPCR) was used to measure relative mRNA
Results. Treatment with resveratrol resulted in growth
suppression and an increase in the cleavage of caspase-3
and PARP. A dose-dependent inhibition of ASCL1, a
neuroedocrine transcription factor, was observed at the
protein and mRNA levels. Protein levels of CgA, a marker
of hormone secretion, were also reduced after treatment
with resveratrol. A dose-dependent induction of Notch2
mRNA was observed by qPCR.
Conclusions. Resveratrol suppresses in vitro growth,
likely through apoptosis, as demonstrated by cleavage of
caspase-3 and PARP. Furthermore, resveratrol decreased
neuroedocrine markers ASCL1 and chromogranin A.
Induction of Notch2 mRNA suggests that this pathway may
be central in the anti-MTC effects observed.
Medullary thyroid cancer (MTC) is derived from the
accounts for 3 to 5% of thyroid cancer cases, although up
to 14% of thyroid cancer deaths.1Although early total
thyroidectomy may be potentially curative for familial
cases of MTC, sporadic MTC accounts for approximately
75% of cases. Surgical resection in the patient population,
however, results in a recurrence rate of almost 50%.
Additionally, patients with MTC experience debilitating
endocrinopathies, related to excessive hormone secretion.
The acidic glycopeptide chromogranin A (CgA) is typi-
cally co-secreted with these hormones and is used as a
clinical marker of the disease.2–4Traditional adjunctive
therapies, including chemotherapy and radiotherapy, have
shown limited efficacy, highlighting the need for novel
Although the role of Notch as an oncogene is best
defined in breast, colorectal, prostate, and pancreatic ade-
nocarcinoma, the Notch signaling pathway has been shown
to play a paradoxical tumor-suppressive role in neuroedo-
crine cancer types.10The Notch genes encode transmem-
brane receptors that regulate cellular differentiation,
development, proliferation, and survival in many contexts.
Upon binding of a ligand, a series of proteolytic cleavage
steps occurs, and the Notch intracellular domain is
released. This fragment translocates to the nucleus to
activate a variety of target genes. In MTC cells, overex-
pression of Notch1 has been shown to inhibit growth and
suppress achaete-scute complex-like 1 (ASCL1), an
important helix-loop-helix transcription factor that regu-
lates the neuroedocrine cancer phenotype and has been
correlated with poor prognosis in neuroedocrine tumors.11
? Society of Surgical Oncology 2010
First Received: 16 August 2010
H. Chen, MD, FACS
Ann Surg Oncol
ASCL1 has been shown to play a particularly important
role in the thyroid: ASCL1-null mice fail to develop par-
afollicular cells.12,13Additionally, through ASCL1, Notch
activation has been shown to suppress the levels of CgA.3,4
Work by our group and others has validated the Notch
pathway as a tumor-suppressing pathway in MTC. Over-
expression studies showed that Notch can suppress growth
as well as ASCL1 expression. Pharmacologic induction of
the Notch pathway was additionally shown to be a potent
anti-MTC strategy in vitro.14–16This finding was subse-
quently confirmed in vivo.17Thus, drugs that target the
Notch pathway are candidates for treatment of MTC.
Recently, our group developed a quantitative high-
throughput screen to identify Notch-activating compounds.
Pinchot et al. identified and validated resveratrol as a
potential Notch activator in gastrointestinal and pulmonary
carcinoid cells. Resveratrol treatment was shown to inhibit
the growth of carcinoid cells both in vitro and in vivo
while suppressing the expression of ASCL1, CgA, and
Resveratrol is a dietary polyphenol found in the skins of
grapes and peanuts. A growing body of evidence suggests
that resveratrol may delay the onset of a variety of ill-
nesses, including cancer, cardiovascular disease, and
ischemic injuries.19High doses of resveratrol in vivo did
not reveal any harmful alterations in hematology, clinical
chemistry, and organ histopathology.20Currently, phase I/
II clinical trials of resveratrol are underway for the treat-
ment of colorectal cancer and lymphoma.21
In this study, we extended our analysis of resveratrol
from carcinoid to MTC. We show that resveratrol sup-
presses growth, induces apoptosis, reduces ASCL1 and
CgA expression, and increases Notch2 mRNA in MTC
cells. To our knowledge, these results are the first
description of a Notch2 activator in MTC and suggest that
resveratrol may be a potential therapeutic option for MTC.
Human MTC (TT) cells were obtained from American
Type Culture Collection (Manassas, VA) and maintained in
RPMI 1640 (Invitrogen Life Technologies, Carlsbad, CA)
supplemented with 16% fetal bovine serum (Sigma),
100 IU/ml penicillin, and 100 lg/ml streptomycin in a
humidified atmosphere of 5% CO2in air at 37?C.
Cell Proliferation Assay
TT cell proliferation was measured by MTT (3-[4,5-
dimethylthiazol-2-yl]-2,5 diphenyl tetrazolium bromide)
assay as previously described.13Cells were seeded in
quadruplicate into 24-well plates and were incubated
overnight to allow cell adhesion. After incubation, cells
were treated with 0 to 25 lM resveratrol (Biomol Inter-
national, Plymouth Meeting, PA). Treatment medium was
changed every 2 days with new dilutions of drug. To per-
form the MTT assay, cells were washed with phosphate-
buffered saline and incubated in 250 ll of serum-free
RPMI 1640 containing 0.5 mg/ml MTT for 4 h. After
incubation, 750 ll of dimethyl sulfoxide (Fisher Scientific,
Pittsburgh, PA) was added to each well and mixed thor-
oughly. Absorbance at 540 nm was measured with a
spectrophotometer (lQuant; Bio-Tek Instruments, Winoo-
ski, VT) and plotted as an average ± standard error of the
TT cells were incubated overnight to allow cell adhe-
sion. After incubation, cells were treated for 2 days with
varying doses of resveratrol. Total protein was collected as
previously described and quantified with the BCA Protein
Assay Kit (Thermo Scientific, Waltham, MA) according to
extracts were resolved on a 10% sodium dodecyl sulfate–
polyacrylamide gel electrophoresis gel, transferred onto
nitrocellulose membranes (Bio-Rad Laboratories, Hercu-
les, CA), and blocked in milk. Membranes were incubated
overnight in primary antibodies with the following dilu-
tions: 1:2000 for mammalian achaete scute homologue-1
for ASCL1 (BD PharMingen, San Diego, CA); 1:500 for
CgA (Zymed Laboratories, San Francisco, CA); 1:1000 for
poly-ADP ribose polymerase (PARP), caspase 3, and
cleaved caspase 3 (Cell Signaling Technology, Beverly,
MA); and 1:10,000 for glyceraldehyde-3-phosphate dehy-
Membranes were then incubated in appropriate amounts of
horseradish peroxidase–conjugated goat anti-mouse or
anti-rabbit antibody (Cell Signaling Technology), which
were detected by Immun-star (Bio-Rad) or Supersignal
West Femto (Pierce Protein Research Products, Rockford,
IL) kits, according to the manufacturer’s instructions.
Quantitative Real-Time Polymerase Chain Reaction
To quantify mRNA expression, we used quantitative
real-time polymerase chain reaction (qPCR). After treat-
ment as above, total RNA was isolated with the RNeasy
Mini kit (Qiagen, Valencia, CA) according to the manu-
facturer’s instructions. The iScript cDNA Synthesis Kit
(Bio-Rad) was used to synthesize cDNA using 2 lg of
RNA per sample. Amplification and detection of PCR
products were performed with iQ SYBR Green (Bio-Rad).
M. Truong et al.
The following PCR primer pairs were used: Notch2
GCACTGACAGTAAT-30), ASCL1 (50-TCC CCC AAC
TAC TCC AAC GAC-30and 50-CCC TCC CAA CGC
CAC TG-30), and GAPDH (50-ACCTGCCAAATATGAT
following conditions were used for PCR amplification:
95?C for 3 min; 35 cycles of: 95? for 30 s, 60? for 25 s, 72?
for 30 s; then 95? for 1 min and 55? for 1 min. All PCR
reactions were performed in triplicate. Threshold cycles
(Ct) were measured with the iCycler Real-Time PCR
Instrument and iCycler Software (Bio-Rad). Target gene
expression levels were calculated by the DCt method with
the formula 2(Ct(GAPDH)–Ct(target)), as described in the
Real-Time PCR Applications Guide (Bio-Rad). Expression
in treatment groups was compared to control expression
and plotted as an average ± standard error of the mean.
Statistical analysis was conducted by one-way analysis
of variance (SPSS software, version 10.0; SPSS, Chicago,
IL). A P value of \0.05 was considered statistically
Resveratrol Inhibits MTC Proliferation
Human MTC cells were treated for up to 6 days with
increasing doses of resveratrol and the MTT assay per-
formed after 4 and 6 days of treatment. Compared to
control, 25, 50, and 100 lM resveratrol treatments for
4 days reduced growth by 5, 8.9, and 16.4%, respectively
(P\0.05). Treatment for 6 days resulted in a 19, 38, and
38% suppression of growth, respectively (P\0.002)
Resveratrol Induces Cleavage of Apoptotic Markers
To explore the mechanism of resveratrol-induced
growth suppression, we examined cleaved caspase 3 and
cleaved poly-ADP ribose polymerase (cl-PARP) by Wes-
tern blot testing. The intrinsic and extrinsic apoptotic
pathways both result in the cleavage of caspase 3, a critical
protease that cleaves other apoptotic proteins such as poly-
ADP ribose polymerase (PARP) to promote cell death.
Thus, increased cleavage of caspase 3 and PARP suggest
activation of apoptotic pathways.22–24We found that
treatment with resveratrol resulted in increased levels of
both cleaved caspase 3 and cl-PARP. We also probed for
full-length caspase 3 and PARP to verify that increased
cleaved caspase 3 and cl-PARP are attributable to proteo-
lytic cleavage, as opposed to an increase in total protein
expression (Fig. 2).
Resveratrol Suppresses Markers of MTC
We hypothesized that MTC growth inhibition would be
associated with a change in neuroedocrine markers. Thus,
we examined levels of ASCL1 and CgA, both important
markers of MTC.2–4,11By means of Western blot testing,
FIG. 1 Resveratrol inhibits medullary thyroid cancer (MTC) cell
proliferation. Human MTC (TT) cells were treated with increasing
doses of resveratrol for up to 6 days, and cell viability was measured
using the MTT (3-[4,5-dimethylthiazol-2-yl]-2,5 diphenyl tetrazolium
bromide) assay. A dose-dependent, statistically significant reduction
in MTC viability was observed (P\0.05)
FIG. 2 Resveratrol treatment induces markers of apoptosis in
medullary thyroid cancer (MTC) cells. Human MTC (TT) cells were
treated with resveratrol for 2 days. Cell lysates were prepared and
probed for apoptotic markers by Western blot testing. Cleavage of
poly-ADP ribose polymerase (PARP) was observed with resveratrol
treatment. Additionally, resveratrol caused a dose-dependent increase
in the cleavage of caspase 3. GAPDH (glyceraldehyde-3-phosphate
dehydrogenase) is presented as a loading control
Resveratrol Induces Apoptosis in MTC
we found that treatment of resveratrol resulted in a dose-
dependent reduction in CgA after 2 days of treatment
(Fig. 3a). This finding is notable because CgA is co-
secreted with bioactive hormones and used as a clinical
marker of disease.2,4
ASCL1 is transcription factor that is highly expressed in
MTC and is strongly correlated with levels of CgA.13
Furthermore, elevated ASCL1 protein is associated with
poor prognosis in related neuroendocrine tumors.25,26Thus,
we performed Western blot analysis for ASCL1 and found
that resveratrol suppressed ASCL1 protein levels after
2 days of treatment (Fig. 3a).
Because ASCL1 suppression could occur at a number of
levels, we performed qPCR for ASCL1 mRNA. Treatment
with resveratrol resulted in a dose-dependent inhibition of
ASCL1 mRNA levels. Notably, a 70% reduction in ASCL1
mRNA levels was achieved with 100 lM treatment of
resveratrol (P\0.001) (Fig. 3b).
Resveratrol Induces Notch2 mRNA
Previous studies have attributed growth inhibition,
induction of apoptosis, and suppression of MTC markers to
Notch pathway activation.27Thus, we investigated the
effects of resveratrol on Notch2 mRNA expression by
qPCR. Treatment with resveratrol resulted in a dose-
dependent induction of Notch2 mRNA expression, with a
significant 15-fold induction with the 100 lM dose
(P\0.001) (Fig. 4). We also examined mRNA expression
of other Notch isoforms and did not observe an induction
(data not shown).
MTC is more difficult to treat and results in more deaths
than other forms of thyroid cancers.28In addition to a high
mortality rate, MTC is associated with debilitating symp-
toms such as dyspnea, dysphagia, diarrhea, and flushing,
associated with increased levels of hormone secretion and
CgA.2,10Because surgery is the only curative option and
traditional chemotherapy and radiotherapy have limited
efficacy, novel therapeutic approaches are necessary.
The Notch1 pathway has been validated as a tumor sup-
pressor in MTC and has been shownto suppress ASCL1 and
CgA expression.13Previous studies by our group and others
seems to be marked overlap in the molecular targets of
Notch1 and Notch2.29Recently, Notch2 has been shown to
FIG. 3 Resveratrol suppresses markers of medullary thyroid cancer
(MTC). a Resveratrol inhibits Achaete-Scute Complex-Like 1
(ASCL1) and chromogranin A (CgA) protein expression. Treatment
for 2 days with increasing doses of resveratrol suppressed the protein
expression of ASCL1 and CgA, two well-known markers of MTC.
GAPDH (glyceraldehyde-3-phosphate dehydrogenase) is presented as
a loading control. b Resveratrol inhibits ASCL1 mRNA expression.
Treatment for 2 days with resveratrol resulted in the dose-dependent
suppression of ASCL1 mRNA expression. Notably, a 70% reduction
in mRNA expression was observed with 100 lM treatment of
resveratrol (P\0.001). Expression levels were normalized to
GAPDH from the respective samples and plotted vs. control
FIG. 4 Resveratrol induces Notch2 mRNA expression. Medullary
thyroid cancer (MTC) cells were treated for 2 days with increasing
doses of resveratrol and Notch2 mRNA expression analyzed by
quantitative real-time polymerase chain reaction. A dose-dependent
induction in the mRNA level of this important tumor suppressor was
observed (P\0.001). Expression levels were normalized to GAPDH
(glyceraldehyde-3-phosphate dehydrogenase) from the respective
samples and plotted vs. control expression
M. Truong et al.
induce many of the same biological effects as Notch1 in
tumors of neuroedocrine origin. In gastrointestinal and pul-
monary carcinoid cells, blocking Notch2 with small
interfering RNA has been shown to rescue the resveratrol-
induced ASCL1 suppression.18We extend our group’s pre-
vious work with resveratrol here by examining the effects of
resveratrol in MTC.
Our study showed that resveratrol is able to greatly
suppress the in vitro proliferation of MTC cells after just
4 days of treatment. Although the degree of growth sup-
pression was modest, the effect on neuroendocrine markers
was substantial. Additionally, we demonstrate an increased
cleavage in apoptotic markers, suggesting that induction of
apoptosis is the mechanism of growth inhibition. Western
blot analysis revealed suppression of CgA, a marker of
hormone secretion. These data suggest the resveratrol is
capable of suppressing bioactive hormones implicated in
the debilitating endocrinopathies associated with MTC. We
show that resveratrol suppresses the protein and mRNA
levels of ASCL1, a transcription factor that is critical in
normal parafollicular cell development. ASCL1 is also
correlated with poor prognosis in neuroendocrine tumors
and isaknown mediator
MTC.1,4,10,12,13,30We additionally show that these changes
are associated with an induction of Notch2, a tumor-sup-
pressing pathway in MTC.
In this study, we demonstrate the anti-MTC potential of
resveratrol and provide what is to our knowledge the first
description of a pharmacologic Notch2-inducing com-
pound in MTC. Resveratrol is a naturally occurring
compound with an established safety profile.19–21Given
the demonstrated efficacy in vitro and known in vivo safety
profile, resveratrol is worthy of additional preclinical
Surgery T35 Short Term Training Grant (DK 062709-0401 to M.T.),
Howard Hughes Medical Institute (to M.R.C.), National Institutes of
Health (Grants RO1 CA121115 and RO1 CA109053 to H.C.), and
American College of Surgeons George H. A. Clowes Jr. Memorial
Research Career Development Award (to H.C.), and Carcinoid Can-
cer Foundation Research Award (to H.C.).
Supported in part by Department of
CONFLICTS OF INTEREST
The authors declare no conflict of
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