Autophagy: A new target for advanced
papillary thyroid cancer therapy
Chi-Iou Lin, PhD,aEdward E. Whang, MD,aMichael A. Abramson, MD,aXiaofeng Jiang, PhD,b
Brendan D. Price, PhD,bDavid B. Donner, PhD,cFrancis D. Moore, Jr, MD,aand
Daniel T. Ruan, MD,aBoston, MA, and San Francisco, CA
Background. Autophagy is a conserved response to stress that facilitates cell survival in some contexts
and promotes cell death in others. We sought to characterize autophagy in papillary thyroid cancer
(PTC), and to determine the effects of autophagy inhibition on chemosensitivity and radiosensitivity.
Methods. The human thyroid papillary carcinoma cell lines TPC-1 and 8505-C were treated with
doxorubicin or radiation in the presence or absence of the autophagy-specific inhibitor 3-methyladenine
Results. Although light chain 3 (LC3)-II protein levels were undetectable in normal thyroid and PTC
specimens at baseline, doxorubicin exposure induced LC3-II expression and the formation of
autophagosomes. Both PTC cell lines expressed low levels of LC3-II under standard conditions.
Treatment of these cells with doxorubicin strongly induced LC3-II expression and the formation of
autophagosomes; however, doxorubicin--mediated induction of LC3-II was abrogated by 3-MA.
Moreover, 3-MA significantly increased the doxorubicin IC50in both PTC cell lines. Radiation exposure
also induced LC3-II expression. Treatment with 3-MA abrogated the radiation--induced increase in
LC3-II in both cell lines and reduced radiosensitivity by 49% and 31% in 8505-C and TPC-1 cells,
Conclusion. Autophagy inhibition promotes PTC resistance to doxorubicin and radiation. Therefore,
autophagy activation may be a useful adjunct treatment for patients with PTC that is refractory to
conventional therapy. (Surgery 2009;146:1208-14.)
From the Department of Surgery, Brigham and Women’s Hospital,aBoston; Department of Radiation
Oncology, Dana-Farber Cancer Institute,bBoston, MA; Department of Surgery, University of California at
San Francisco,cSan Francisco, CA
THYROID CANCER represents 90% of all endocrine
(PTC) accounting for 80% of all thyroid cancers.2
Whereas PTC tends to have a favorable prognosis
overall, a subset of PTC is refractory to surgery
and radioactive iodine ablation.3Patients with
advanced PTC have been treated with external
beam radiation and doxorubicin, which is the
only chemotherapeutic agent approved by the
Food & Drug Administration for thyroid cancers
refractory to radioactive iodine. Unfortunately,
PTC is highly resistant to external beam radiation
and doxorubicin; novel therapies are needed for
patients with advanced disease.4
Autophagy is a catabolic process by which
damaged or long-lived cellular proteins and
organelles are degraded.5A range of stressors
can induce autophagy, which is associated with
the formation of an autophagosome, a double-
membrane vesicle that engulfs organelles and cyto-
plasm, and subsequent lysosomal degradation of
this structure.6,7In some cancer cells, autophagy
has been reported to play a protective role under
conditions of stress8; however, prolonged autoph-
agy in cancer cells that lack the machinery to
undergo apoptosis can result in a nonapoptotic
programmed cell death.9As such, it is still unclear
if autophagy has primarily a protective or destruc-
tive effect on cancer cells.
There is accumulating evidence that both
external radiation10and chemotherapy agents,
like doxorubicin11,12can induce autophagy in
cancer cells in vitro. Furthermore, a recent study
demonstrated that the chemotherapy agent, com-
bretastatin A4 phosphate, induces autophagy in a
nude mouse xenograft model of anaplastic thyroid
Supported by research grants from the Department of Surgery,
Brigham and Women’s Hospital, Boston, MA.
Presented at the 30th Annual Meeting of the American Associ-
ation of Endocrine Surgeons, Madison, Wisconsin, May 2--5,
Reprint requests: Daniel T. Ruan, MD, Department of Surgery,
Brigham and Women’s Hospital, 75 Francis Street, Boston,
MA 02115. E-mail: email@example.com.
0039-6060/$ - see front matter
? 2009 Mosby, Inc. All rights reserved.
cancer.13To date, however, no studies have evalu-
ated the effects of doxorubicin or radiation on
the autophagic activity of PTC cells. As such, in
the present study, we investigated the effects of
doxorubicin and radiation on autophagy in PTC
cells. Furthermore, we evaluated the impact of
autophagy inhibition on PTC sensitivity to doxoru-
bicin and radiation.
Materials. Microtubule-associated protein 1 light
chain 3 (LC3) immunoblotting is a technique used
the protein expression level serves as a good indica-
tor of autophagic activation.15Mouse anti-human
LC3 antibody was purchased from MBL Interna-
tional (Nagoya, Japan). Mouse anti-actin antibody
CA). Secondary horseradish peroxidase-conjugated
anti-mouse antibody was obtained from Vector Lab-
oratories (Burlingame, CA). 3-Methyladenine (3-
MA), which is a widely used inhibitor of autophagy
pathway of degradation,16was purchased from Cal-
biochem (La Jolla, CA). Doxorubicin was obtained
from Fluka (Ronkonkoma, NY).
Human thyroid tumor tissue specimens. Human
obtained from thyroidectomy specimens resected
from surgery patients. The specimens were frozen
immediately in liquid nitrogen on removal from
the patients, and the samples were stored frozen
until the time of protein extraction. Tissue lysates
were prepared using radioimmunoprecipitation
assay (RIPA) buffer containing protease inhibitor
cocktail, as described previously.17This protocol
was approved by the institutional review board of
Brigham and Women’s Hospital, Boston, MA.
Ex vivo PTC tumor specimen culture. A piece of
each tissue specimen was excised immediately after
thyroidectomy using traditional sterile techniques.
Vital specimens (0.5 mm3) were cultured in cell
culture inserts (Millicell; Millipore, Billerica, MA)
containing 600 mL of Dulbecco’s modified Eagle’s
medium: F12 (DMEM/F12) with 15 mM of HEPES
(Sigma-Aldrich, St Louis, MO); this was supple-
mented with GlutaMAX-I (Invitrogen; Carlsbad,
CA). The specimens were cultured at 37?C and
5% CO2for 8 hours in the absence or presence
of doxorubicin (1 mM).
Cell culture. The human thyroid papillary carci-
noma cell lines TPC-1 and 8505-C were cultured, as
describedpreviously.18,19TPC-1 and 8505-Cwere ob-
tained from Orlo H. Clark, MD (University of
California at San Francisco, San Francisco, CA),
and Sareh Parangi, MD (Massachusetts General
Hospital, Boston, MA), respectively. Cells were
subcultured at 80% confluency by trypsinization in
a 0.5% (v/v) trypsin solution, supplemented with
Western blotting. Cell lysates were separated on
4%;12% sodium dodecyl sulfate polyacrylamide
gel electrophoresis (SDS-PAGE) gels and trans-
ferred electrophoretically to a polyvinylidene di-
fluoride (PVDF) membrane (Invitrogen). The
membranes were blocked with 5% casein in a Tris-
buffered saline-Tween 20 solution. The blots were
performed as described previously.17Proteins on
each immunoblot were visualized with a Western
blot chemiluminescence reagent (Amersham Bio-
sciences, Piscataway, NJ).
Electron microscopy. Tissue specimens or cells
were washed with serum-free DMEM (Invitrogen)
with a brief wash in 1 M cacodylate buffer and then
fixed with 2.5% glutaraldehyde with 0.1 M sodium
cacodylate buffer for 1 hour. Fixed cells were de-
tached and then processed at the Harvard Medical
School Electron Microscopy core facility by using
protocols described previously,20using an electron
microscope (60kV; JEOL, Tokyo, Japan).
MTS cell proliferation assay and IC50determi-
2H-tetrazolium) assay (CellTiter 96 AQueous;
Promega, Madison, WI) was used to assess doxo-
rubicin effects on cell viability. Cells were trypsi-
nized, and 5000 cells/well were seeded in a 96-well
plate; 24 hours later, standard culture media were
replaced with doxorubicin-containing media (final
concentrations of 10,000, 5000, 1000, 500, 100, 50,
or 10 nM) or standard culture media without
doxorubicin. After 48 hours of treatment in a
humidified CO2incubator at 37?C, MTS reagent
(20 mL) was added to each well.
After 2 hours, absorbance at 490 nm (A490) was
quantified using a Spectramax M5 Microplate
Response was defined as (mean drug-treated A490
-- blank)/(mean untreated control A490-- blank)
3 100. The data were analyzed by weighted nonlin-
ear regression to determine the IC50, defined as
the doxorubicin concentration that inhibited
MTS metabolism to 50% that of untreated con-
trols. Each dosage was evaluated in triplicate.
Radiation exposure. For radiation experiments,
cells were trypsinized, replated, and incubated for
an additional 24 hours under standard conditions.
Cells were then irradiated with doses ranging from
0 to 6 Gy (EXS-300 X-irradiator; Toshiba, Tokyo,
Volume 146, Number 6
Lin et al 1209
Japan; 200 kV, 15 mA, 1 Gy/min). At 48 hours after
irradiation, cell viability was assayed using MTS as
Statistical analysis. Data were analyzed by 1-way
analysis of variance followed by the unpaired t
test. Each assay was done in triplicate, and a
value of P < .05 was considered statistically
Doxorubicin induces autophagy in freshly har-
vested human PTC specimens. By immunoblot
analysis, LC3-I and -II protein expression was
undetectable in freshly harvested thyroid and
PTC specimens (Fig 1, A); however, exposure of
PTC to doxorubicin for 8 hours induced the
expression of LC3-II protein ex vivo (Fig 1, B). Fur-
thermore, PTC exposed to doxorubicin ex vivo
induced the formation of autophagosomes, which
was evident on electron microscopy (Fig 1, C). This
observation suggests that autophagic activity is low
at baseline in PTC specimens from patients at the
time of thyroidectomy. Treatment with doxorubi-
cin exposure, however, activates autophagy in
human PTC tumors.
Doxorubicin treatment stimulates autophagy,
and this effect is blocked by 3-MA. In both 8505-
C and TPC-1 cells, low levels of LC3-I and LC3-II
were detected by immunoblotting (Fig 2, A). Dox-
orubicin induced LC3-II protein expression in a
dose-dependent manner, indicating the activation
3-MA inhibited the doxorubicin--induced LC3-II
protein accumulation. To confirm these findings,
we performed electron microscopy on 8505-C cells
under the same conditions. Autophagosomes were
not detected in control cells but were abundant in
cells exposed to doxorubicin (Fig 2, B). Further-
more, no autophagosomes were detected on elec-
tron microscopyin 8505-C
pretreated with 3-MA. Similarly, the ex vivo PTC
specimens pretreated with 3-MA showed no induc-
tion of autophagosomes when exposed to doxoru-
bicin (Fig 1, C). Taken together, these results
indicate that doxorubicin upregulates autophagy
in PTC and that this effect can be blocked with
Inhibition of autophagy decreases chemosensi-
tivity to doxorubicin. 8505-C and TPC-1 cells were
pretreated with either dimethyl sulfoxide (DMSO)
cells that were
Fig 1. Endogenous light chain 3 (LC3)-1 and -II expression level in human normal thyroid and papillary thyroid cancer
(PTC) tissue. (A) Fresh human normal thyroid and PTC specimens from 4 consecutive patients were collected. Tissue
lysates were prepared and LC3-I and -II protein expression levels were monitored by Western blotting. Reprobing
against actin was done to ensure equal protein loading. (B) In 3 additional patients, LC3-I and -II protein expression
levels were determined in normal and PTC tissue after exposure to control conditions (dimethyl sulfoxide [DMSO]
only) and in PTC exposed to 8 hours of doxorubicin. (C) Representative electron microscopic images showing autopha-
gosomes after DMSO, doxorubicin (1 mM), doxorubicin (1 mM) + 3-methyladenine (3-MA) (1 mM) treatment for
16 hours in PTC tissue specimens. Arrows depict double-membrane autophagic vacuoles containing recognizable organ-
elles and cellular material. (Original magnification 3 15,000.) DOX, Doxorubicin.
1210 Lin et al
or 1 mM of 3-MA for 1 hour followed by coincuba-
tion with either 3-MA or dimethyl sulfoxide
(DMSO) combined with doxorubicin (0 to 10000
nM) for 48 hours. MTS assays were done, and
IC50 for doxorubicin was determined. 3-MA in-
creased doxorubicin IC50by 33% (96 nM to 127
nM) and 39% (78 nM to 109 nM) in 8505-C and
TPC-1 cells, respectively (Fig 3). These results indi-
cate that autophagy inhibition decreases PTC che-
mosensitivity to doxorubicin.
Radiation induces autophagy in PTC cells. A
total of 4 Gy of external radiation induced LC3-II
protein expression in both 8505-C and TPC-1 cells
(Fig 4). Similar to the previously described experi-
ments with doxorubicin, pretreatment with 3-MA
inhibited the radiation--induced LC3-II expression
in both cell lines. This finding indicates that ioniz-
ing radiation activates autophagy in PTC; this
effect can be blocked with 3-MA.
Inhibition of autophagy decreases radiosensitiv-
ity in PTC cells. 8505-C and TPC-1 cells were
exposed to radiation doses ranging from 0 to 6 Gy.
Viability was measured using MTS 48 hours after
external radiation treatment. Radiation exposure
decreased cellular viability relative to controls in a
dose-dependent manner (Fig 5). Treatment with
3-MA increased the viability of cells treated with
external radiation, indicating that autophagy inhi-
bition promotes radioresistance in PTC.
Autophagy is an evolutionarily conserved pro-
cess that entails the degradation and recycling of
organelles during times of cellular stress.22In the-
ory, autophagy could enable cancer cell survival
during times of cellular stress from chemotherapy
exposure, ionizing radiation, or hypoxia. Thus,
autophagy could protect cancer cells from unfavor-
able tumor microenvironments and promote resis-
tance to cancer therapies.
Autophagy, however, may also promote cancer
cell death. In cells that lack the machinery to
undergo apoptosis, autophagic cell death is an
alternative means of programmed cell death.23
Furthermore, some tumor suppressors induce
autophagy,24and some oncogenic proteins sup-
press autophagy.25In fact, Beclin 1, a component
of the autophagosome, is a tumor suppressor.25,26
Therefore, in some contexts, there is also evidence
indicating that autophagy has anticancer effects.
Our results suggest that autophagy is not
dysregulated in PTC, because both normal thyroid
tissue and PTC demonstrate low levels of autopha-
gicactivityat the timeof thyroidectomy.
Fig 2. Effects of doxorubicin on autophagy induction in 8505-C and TPC-1 cells. (A) 8505-C or TPC-1 cells were pre-
treated with or without 1 mM of 3-methyladenine (3-MA) for 16 hours followed by treatment with control (dimethyl sulf-
oxide [DMSO]), 1 or 4 mM of doxorubicin, or 4 mM of doxorubicin with 1 mM of 3-MA for 48 hours, as indicated . Total
cell lysates were isolated and light chain 3 (LC3)-I and -II protein expression levels were monitored by Western blotting
using an antihuman LC3 antibody. Reprobing against actin was done to ensure equal protein. (B) Representative elec-
tron microscopic images showing autophagosomes after DMSO, doxorubicin (1 mM), doxorubicin (1 mM) + 3-MA
(1 mM) treatment for 16 hours in 8505-C cells. Arrows depict double-membrane autophagic vacuoles containing recog-
nizable organelles and cellular material. (Original magnification 3 15,000.) Dox, Doxorubicin.
Volume 146, Number 6
Lin et al 1211
Furthermore, autophagy is activated briskly in PTC
from exposure to either doxorubicin or external
beam radiation, indicating that autophagic activity
is preserved in PTC. Lastly, we found that the
inhibition of autophagy decreased the tumor cell
response to both doxorubicin and external beam
Doxorubicin is an anthracycline antibiotic that
induces DNA damage and apoptosis in cancer
cells.27Doxorubicin can activate autophagy in can-
cer cells.11,12Furthermore, autophagy inhibition
protects against doxorubicin--induced cell death
in HeLa and cervical cancer cells cell lines.28
Before this report, the influence of autophagic
Fig 3. Effects of 3-methyladenine (3-MA) on chemoresistance to doxorubicin in 8505-C and TPC-1 cells. 8505-C (A) or
TPC-1 cells (B) were pretreated with control (dimethyl sulfoxide [DMSO]) or 1 mM of 3-MA for 16 hours. Cells were
then incubated with different concentrations of doxorubicin with or without 3-MA, and IC50of doxorubicin was deter-
mined by MTS assay. The absorbance of each well is proportional to cell survival, and the absorbance of cells treated
with no doxorubicin was set to 100% survival. The y axis indicates the relative percent survival. The curves show means
± SEM of 3 independent experiments. *P < .05, indicating a significant statistical difference between doxorubicin treat-
ment--decreased survival percent in the presence or absence of 3-MA.
Fig 4. Effects of radiation on autophagy induction in 8505-C and TPC-1 cells. 8505-C or TPC-1 cells were treated with or
without 1 mM of 3-methyladenine (3-MA) for 16 hours and then exposed to 0, 2, 4, or 6 Gy of radiation, as indicated.
Total cell lysates were isolated and light chain 3 (LC3)-I and -II protein expression levels were assayed by Western blot-
ting using an antihuman LC3 antibody. Reprobing against actin was done to ensure equal protein loading.
1212 Lin et al
activity on doxorubicin chemosensitivity in PTC
was unclear. Consistent with previous reports, we
found that the inhibition of autophagy decreased
chemosensitivity in PTC.
Of note, inhibition of autophagy did not change
chemosensitivity at very high concentrations of
doxorubicin. In other cell types, low doses of
doxorubicin induces autophagy, whereas greater
doses activate predominantly apoptosis.29Simi-
larly, we found that doxorubicin exposure activates
autophagy in PTC and that concentrations exceed-
ing 800 nM potently activates apoptosis.17Not sur-
prisingly, autophagy inhibition did not effect
chemosensitivity at very high concentrations of
doxorubicin, because apoptosis is potently acti-
vated at such high doses.
Finally, previous studies have shown that exter-
nal beam radiation activates autophagy in other
cell types.10,30-33Radiation exposure also activates
autophagy in PTC cells. Furthermore, in other can-
cer cell lines, the inhibition of autophagic activity
decreases radiosensitivity,31,32and the promotion
of autophagic activity increases radiosensitivity.30,33
Our results in PTC are consistent with these previ-
In conclusion, this report is the first, to our
knowledge, to characterize autophagy in PTC cells.
Autophagy does not appear to be activated in PTC
at the time of thyroidectomy, but is robustly acti-
vated by exposure to either chemotherapy or
external radiation. Autophagy inhibition promotes
resistance to doxorubicin and external beam radi-
ation in PTC. Taken together, our data suggest that
the use of agents that promote autophagy may be
useful for the treatment of refractory PTC.
We thank Orlo H. Clark, MD (University of California
at San Francisco, San Francisco, CA), and Sareh Parangi,
MD (Massachusetts General Hospital, Boston, MA) for
kindly providing TPC-1 and 8505-C cells, respectively. We
also would like to thank J. Dirk Iglehart, MD (Dana-
Farber Cancer Institute,
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Dr Mark S. Cohen (Kansas City, KS): My question is with
regard to your ex vivo studies that you looked at. It’s
known that the shock of removing a tumor from the
body can cause upregulation of autophagy. Could that
be contributing to what you are seeing in your culture
specimens with ex vivo?
Dr Daniel T. Ruan (Boston, MA): Yes, it does. At the
time of specimen harvest, LC3-II bands are nonexistent
both in normal tissue and in the papillary thyroid cancer
specimens. You can start to detect LC3-II bands after
approximately 8 hours of ex vivo maintenance conditions.
tinue to synthesize DNA, even after 24 hours. This was
proven by BrdU uptake assays. For the purpose of testing
agents in preclinical studies, we utilized the appropriate
negative controls to account for the upregulation of au-
tophagy that reflects the stress of ex vivo culturing, and
we performed the experiments at the 8-hour time point.
Dr Terry C. Lairmore (Temple, TX): RAD 001, an
agent that promotes autophagy, is now in phase 2 clinical
trials for unresectable neuroendocrine tumors of the
pancreas. I’m just interested in how you got interested
in differentiated follicular thyroid cells, and have you
studied this in the neuroendocrine medullary thyroid
carcinoma cell lines?
Dr Daniel T. Ruan (Boston, MA): We were interested
because papillary thyroid cancer makes up the vast ma-
jority of my practice, like many other surgeons. Further-
more, we used our thyroidectomy specimens for ex vivo
modeling. We have not yet modulated autophagy in
other cell lines.
1214 Lin et al