Send Orders for Reprints to reprints@benthamscience.

net
Current Cancer Drug Targets, 2014, 14, 59-69 59

Dimethylaminoparthenolide, A Water Soluble Parthenolide, Suppresses

Lung Tumorigenesis Through Down-Regulating the STAT3 Signaling
Pathway

Jung M. Song1, Xuemin Qian1, Pramod Upadhyayya1, Kwon H. Hong2 and Fekadu Kassie1,3,*

1
Masonic Cancer Center, 2Institue for Therapeutics Discovery and Development, 3College of Veterinary Medicine,
University of Minnesota, Minnesota, USA

Abstract: Lung cancer is the most fatal cancer and development of agents that suppress lung tumorigenesis is a crucial
strategy to reduce mortality related to this disease. In the present study, we showed, using an in vitro model of lung
tumorigenesis, that dimethylamino-parthenolide (DMAPT), a water soluble parthenolide analog, selectively inhibited the
growth and survival of premalignant and malignant cells with minimal effects on parental immortalized cells. These
effects were paralleled by suppression of pSTAT3, Mcl-1 and cyclin D1 and PARP cleavage, suggesting that the anti-
proliferative and apoptotic effects of DMAPT could be mediated, at least in part, via suppression of the STAT3 signaling
pathway. Moreover, in tobacco smoke carcinogen-induced lung tumor bioassay in mice, intranasal instillation of low
doses of DMAPT significantly reduced the overall lung tumor multiplicity by 39%. Interestingly, the drug was
specifically effective (62% reduction) against bigger lung tumors (> 2 mm), which have a higher potential to develop into
lung adenocarcinoma. Western immunoblotting analyses of mouse lung tissues indicated significantly lower level of
pSTAT3 and Mcl-1 in the carcinogen plus DMAPT group relative to the group treated with the carcinogen only. Given
the evidence that STAT3 is activated in more than half of lung cancers and it regulates genes involved in cell
proliferation, survival and angiogenesis, DMAPT is a promising agent for lung cancer chemoprevention in subjects who
are at high risk of developing this devastating disease.
Keywords: Apoptosis, chemoprevention, dimethylaminoparthenolide, intranasal administration, mouse lung tumorigenesis, 4-
(methylnitrosamino)-1-(3-pyridyl)-1-butanone.

INTRODUCTION from the feverfew plant, Tanacetum parthenium. Parthenolide

has been shown to induce anti-proliferative and apoptotic
Lung cancer is the leading cause of cancer death in the
effects against a wide variety of cancer cells, including
USA and the world. According to the National Cancer
prostate, breast, and pancreatic cancer cells as well as those
Institute’s report, there were 226,160 new lung cancer cases
from hepatoma, leukemia and multiple myeloma, via
and 160,340 lung-cancer related deaths by the end of 2012
inhibition of NF-
B, STAT3, and enhancement of reactive
[1]. Despite significant advances in the treatment of lung oxygen species [12-15]. Nevertheless, this agent has a major
cancer during the last decades, the mortality and morbidity
limitation to be a clinically effective chemopreventive agent
of this disease have not been reduced.
due to its poor water solubility and low bioavailability [16].
Chemoprevention, the use of natural dietary products or This has led to the development of an aminoanalog of
pharmaceutical compounds to suppress the carcinogenesis parthenolide, dimethylaminoparthenolide (DMAPT), which
progress in its early stages, is a promising approach to has a 1000-fold higher water solubility as compared to
reduce cancer-related mortality. This strategy has been found parthenolide [17]. Besides its potent anti-cancer activity and
to be effective in populations at high risk for prostate, colon high bioavailability, DMAPT is also minimally toxic toward
and breast cancers [2-4]. Although various natural and normal cells [18].
synthetic agents have been studied for their chemopreventive
In this study, we examined the in vitro and in vivo
effects against lung cancer [5-11], at present, there are no
efficacy of DMAPT to suppress lung tumorigenesis. In in
clinically effective agents. Therefore, development of novel
vitro assays in which we used a set of bronchial cell lines
chemopreventive agents with high efficacy and minimal with different stages of cell transformation (immortalized,
toxicity is highly required.
premalignant and malignant), DMAPT caused stronger
One promising chemopreventive agent against lung antiproliferative and apoptotic effects in premalignant and
cancer is parthenolide, a sesquiterpene lactone extracted malignant cells than in immortalized parental cells. These
effects were mediated, at least partly, via suppression of
STAT3 activation and expression of Mcl-1, one of the
*Address correspondence to this author at the Masonic Cancer Center, downstream targets of STAT3. In mouse lung tumor
University of Minnesota, Mayo Mail Code 806, 420 Delaware Street SE, bioassays in which lung tumors were induced by the tobacco
Minneapolis, MN 55455, USA; Tel: 612-625-9637; Fax: 612-626-5135; smoke carcinogen 4-(methylnitrosamino)-1-(3-pyridyl)-1-
E-mail: kassi012@umn.edu

1873-5576/14 $58.00+.00 © 2014 Bentham Science Publishers

60 Current Cancer Drug Targets, 2014, Vol. 14, No. 1 Song et al.

butanone (NNK), DMAPT significantly reduced the number culture media were aspirated, 100 μl of dimethyl sulfoxide
and size of lung tumors. Moreover, DMAPT suppressed levels was added to each well and absorbance was read at 570 nm
of pSTAT3 and Mcl-1 in lung tissues of NNK-treated mice. with a plate reader. Each treatment with PBS or DMAPT
was carried out in triplicate and the assays were repeated
MATERIALS AND METHODS three times on different days.
Chemicals, Reagents and Diets
Annexin V/Propidium Iodide Apoptosis Assay
DMAPT was synthesized in two steps from the natural
To determine the apoptotic effects of DMAPT in BEAS-
product parthenolide (LKT laboratories, Minneapolis, MN)
2B and 1170 cell lines, cells were treated with different
as previously described [19]. The chemical structure of
concentrations of DMAPT (5-20 μM) for 48 h. Subsequently,
DMAPT is depicted in Fig. 1. Anti-phospho-STAT3, anti- 1  106 cells were washed twice with cold phosphate-buffered
total STAT3, anti-phospho-Akt, anti-total Akt, anti-phospho-
saline and stained with 5 μl Annexin V-fluorescein
extracellular signal-regulated kinase (ERK), anti-total ERK,
isothiocyanate (BD Pharmingen, San Diego, CA) and 10 μl
anti-phospho-JNK, anti-Bcl-2, anti-MMP9, anti-cyclin D1,
PI (5 μg/ml, BD Pharmingen, San Diego, CA) in binding
anti-survivin, anti-p-p65, anti-p65, anti-IKK, anti-IKB,
buffer [10 mmol/l N-2-hydroxyethylpiperazine-N-2-ethan-
anti--actin and goat anti-rabbit IgG secondary antibody
esulfonic acid (pH 7.4), 140 mmol/l NaOH, and 2.5 mmol/l
were from Cell Signaling Technology (Beverly, MA). Anti- CaCl2] for 15 min at room temperature in the dark. The
poly (ADP-ribose) polymerase (PARP) and anti-Mcl-1 were
apoptotic cells were determined using a Becton Dickinson
obtained from Santa Cruz Biotechnology. Mouse diets (AIN-
FACScan cytofluorometer. Both early apoptotic (Annexin
93G and AIN-93M) were purchased from Harlan Teklad
V-positive, PI-negative) and late apoptotic (Annexin V-
(Madison, WI). The AIN-93G diet, high in protein and fat,
positive and PI-positive) cells were included in cell death
was used to support rapid growth of the mice until eight
determinations. The assay was carried out three times.
weeks of age. AIN-93G diet was then replaced by AIN-93M
diet, a low-protein and low-fat diet, which is recommended Western Blot Analysis
for adult maintenance [20]. AIN-93 diets are standard diets
for lung tumorigenesis studies in A/J mice. For the preparation of cell lysates from the cell lines,
bronchial cells (1  106) treated with DMAPT at different
Cells and Cell Culture concentrations for various time periods were harvested and
incubated in RIPA buffer with protease inhibitor and
Immortalized bronchial epithelial cell line 2B (BEAS-
phophatase inhibitor (Pierce, Rockford, IL) for 10 min on
2B) and its premalignant (1799 and 1198) and malignant
ice. Subsequently, the preparations were centrifuged (14,000
(1170) derivatives were kindly provided by Dr. Klein-Szanto
g for 25 min at 4°C), the supernatants collected, the protein
(Fox Chaser Cancer Center, Philadelphia). Cell line 1799
concentration measured using a BCA protein assay kit
was developed from BEAS-2B cells explanted along with
(Pierce, Rockford, IL), samples aliquoted and stored at
beeswax pellets into rat tracheas that had been denuded of
80°C. For the preparation of mouse lung tissue lysates, lung
bronchial epithelium and further transplanted into the dorsal
tissues from individual mouse (three mice/group) were
subcutaneous tissues of nude mice [21]. Cell lines 1198 and
ground with a mortar and pestle on liquid nitrogen, and the
1170 were developed in a similar manner except that the
powder suspended in ice-cold RIPA lysis buffer for 10 min
beeswax pellets contained cigarette smoke condensate. All
and processed similar to the cell lysates.
bronchial cells were maintained in keratinocyte serum-free
medium with recommended supplements (KSFM; Life For western immunoblotting, aliquots of protein (50 μg
Technologies Inc., Gaithersburg, MD) in a humidified for cell culture sample or 100 μg for lung tissue sample)
atmosphere containing 5% CO2. The cells were of the same were electrophoresed on a 4-12 % Novex Tris–glycine gel
passage number when used for the study. (Invitrogen, Carlsbad, CA), and transferred to a polyvinylidene
difluoride (PVDF) membrane (Bio-Rad). After blocking
Cell Proliferation Assay with tris-buffered saline (TBS) containing 0.05% Tween20
Cell proliferation was determined using the methyl- (TBST) and 5% non-fat powdered milk, the membrane was
incubated with the primary antibody solution at 4oC overnight.
thiazoletetrazolium (MTT; Biotium, Hayward, CA) assay as
Subsequently, the membrane was washed with TBST and
follows. BEAS-2B cells and its three derivatives were plated
incubated with the appropriate horseradish peroxidase-
on a 96-well plate at a density of 6,000 cells/well, grown for
conjugated secondary antibody for 1 h at room temperature.
24 h and treated with DMAPT (0-20 μM) for 48 h followed
The protein-antibody complexes were detected by enhanced
by MTT treatment (100 μl per well) for 4 h. Subsequently,
chemiluminescence (ECL kit) in accordance with the
manufacturer’s directions (Pierce, Rockford, IL). All
membranes were stripped and reprobed with anti--actin
(1:1000) to check for differences in the amount of protein
loaded in each lane. For each protein, at least three western
assays were carried out. For quantitative determination of
protein levels, densitometric measurements of Western blot
bands were performed using digitalized scientific software
program UN-SCAN-IT software (Silk Scientific, Orem,
Fig. (1). Chemical structure of dimethylaminoparthenolide (DMAPT). Utah).
Lung Cancer Chemoprevention with DMAPT Current Cancer Drug Targets, 2014, Vol. 14, No. 1 61

STAT3- and NFkB- DNA Binding Assay a similar manner. At week 8 of the study, the diet was
changed from AIN-93G to AIN-93M. Diet consumption was
To determine inhibition by DMAPT of STAT3 and
measured weekly, and body weights were determined every
NFkB binding to DNA, we used the TransAM transcription
week through out the study. At the end of the study, the mice
factor assay (Active Motif, CA), a non-radioactive
were euthanized by an overdose of carbon dioxide.
transcription factor ELISA kit that facilitate the study of Subsequently, the lungs were harvested and tumors on the
transcription factor activation, and the Odyssey Infrared
surface of the lung counted and their sizes determined under
electrophoretic mobility shift assay (EMSA) Kit (LI-COR,
a dissecting microscope. Lung tissues used for Western blot
Lincoln, NB), which is an excellent alternative method to
studies were stored at 80°C until used in the assay.
radioisotopic and chemiluminescent EMSA detection methods.
For the quantitative analyses of STAT3 and NFkB Statistical Analyses
binding to DNA using the TransAM transcription factor Wilcoxon rank sum test was used for pair-wise
assay (Active Motif, CA), 10 g of nuclear protein extract comparisons of the number of tumors on the surface of the
was diluted in complete lysis buffer and added into each well lung (groups treated with NNK and received DMAPT versus
coated with oligonucleotide containing STAT3 or NFkB the group treated with NNK only). Two-sided p values

consensus binding site. Under the assay conditions, STAT3 0.05 were considered statistically significant. For the
or NFkB subunit proteins in nuclear extract bind to the analyses of the number of tumors and tumor sizes, the two-
oligonucleotide, and STAT3 or p65 was detected by using a sided Student`s t-test was conducted in SAS 9.1.3. Results of
primary antibody specific to the proteins followed by MTT, apoptosis, DNA binding and Western assays were
incubation with horseradish peroxidase conjugated secondary performed using 2-tailed t-test with Graphpad Prism 4
antibody and colorimetric reading at 450 nm. Wild type software (Graphpad, La Jolla, CA). Data are reported as
consensus oligonucleotide (CO) was used as a competitor to mean ± standard deviation of triplicate determinations. * p <
prevent STAT3 or NFkB binding to the probe immobilized 0.05, compared to control.
on the plate. In order to monitor the specificity of the assay,
nuclear protein extracts (5 g) from HepG2 cells stimulated RESULTS
with IL6 (provided in the kit) were used as positive control
samples. Two independent experiments were performed with Effects of DMAPT on the Growth of Immortalized
duplicate samples. Error bars represent standard deviation. (BEAS-2B), Premalignant (1799 and 1198) and Malignant
(1170) Bronchial Cells
For the Odyssey Infrared EMSA assay, first, BEAS-2B
and 1170 cells were treated with different concentrations of In order to assess if DMAPT induces differential anti-
DMAPT for 48 h and nuclear protein was extracted using proliferative activities in bronchial cells at different stages of
extraction kit (Active Motif, CA). Protein concentration was transformation, parental BEAS-2B cell line and its
determined by BCA protein assay kit from Pierce (Rockford, premalignant or malignant derivatives were treated with PBS
IL). Nuclear protein extract (2.5 μg) was incubated with or the test agent for 48 h and cell proliferation rate was
STAT3 IRDye™ 700 infrared dye-labeled oligonucleotides determined by the MTT assay. As shown in Fig. 2, DMAPT
or NFkB IRDye™ 700 infrared dye-labeled oligonucleotides reduced the proliferation of all cell lines in a concentration-
(LI-COR) for 30 min at room temperature. In competition dependant manner. However, the effects of DMAPT on
assay, unlabelled competitor oligonucleotide was added in BEAS-2B cells were significant only at higher concentrations
the assay mixture before the addition of dye-labeled STAT3 of the compound (10 and 20 M), while the proliferation of
or NFkB oligonucleotide. The incubation mixture was 1799, 1198 and 1170 cells was significantly reduced at all
applied to 4-12% TBE gel (Invitrogen) and electrophoresis concentration levels. Therefore, these results indicate that
was performed at 10 V/cm at 4°C. The gels were scanned lower concentrations of DMAPT (< 10 M) could be used to
with the Odyssey scan bed (LI-COR). block the growth of premalignant and malignant bronchial
cells with only minimal effects on normal cells.
Tumor Bioassay
DMAPT Causes Differential Apoptotic Effects in
Female A/J mice (6 weeks old) were obtained from The Malignant bronchial cells Versus Immortalized Cells
Jackson Laboratory (Bar Harbor, ME). Upon arrival, the
mice were housed in the specific-pathogen-free animal Following the observation that DMAPT caused
quarters of Research Animal Resources, University of differential anti-proliferative effects in premalignant and
Minnesota Academic Health Center, randomized into three malignant cells, we sought to compare the apoptotic effects
groups and maintained on pelleted AIN-93G diet. One week of the agent in immortalized BEAS-2B cells versus malignant
after arrival, the mice were switched to AIN-93G-powdered 1170 cells. As depicted in Fig. 3, DMAPT increased
diet and treated with NNK (two doses of 50 mg/kg, once a the percentage of apoptotic 1170 cells in a concentration-
week, in 0.3 ml physiological saline solution) or the vehicle dependent manner and these effects were significant,
by intraperitoneal injection. Beginning one week after the compared to the rate of apoptosis in PBS-treated cells, at all
last dose of NNK, mice received intranasal instillation of concentrations tested except at 5 M of DMAPT. When the
DMAPT (10 mg/kg, in 50 μl physiological saline solution), apoptotic cells were grouped as early apoptotic cells
twice weekly, until the termination of the study at week 16. (annexin V+/PI) and late apoptotic cells (annexin V+/PI+)
This dose level of DMAPT is more than 20-fold lower than (data not shown), it became clear that low concentrations of
the dose of the agent administered orally to mice in earlier DMAPT (7.5, 10 M) predominantly induce early apoptotic
studies [17]. Control mice were treated with the vehicle in cells, whereas higher concentrations of DMAPT (15, 20 M)
62 Current Cancer Drug Targets, 2014, Vol. 14, No. 1 Song et al.

DMAPT Down-regulates pSTAT3 and Related Proteins

in 1170 Cells in a Concentration-dependent Manner, but
not in Parental Immortalized BEAS-2B Cells
Earlier studies in various cancer cells showed that the
most important molecular targets of parthenolide, the parent
compound for DMAPT, are the NF-
B and STAT3 signaling

Fig. (2). Effect of DMAPT on the proliferation of parental immortalized

human bronchial BEAS-2B cell line and its premalignant (1799, 1198)
and malignant (1170) derivatives. Cells were grown on 96-well plates
for 24 h and exposed to DMAPT (5, 7.5, 10 or 20 μM) for 48 h and cell
proliferation was determined by MTT assay. The results were presented
as mean ± SD of the percentage of the control. *P < 0.05.

Fig. (3). DMAPT induced concentration-dependent apoptotic activities

in 1170 cells, but only minimal effects in BEAS-2B cells. Cells were
exposed to DMAPT (5-20 μM) for 48 h and the frequency of apoptotic
cells was determined by FACS analysis after Annexin V/PI-staining.
The data shown are mean ± SD from three studies. *P < 0.05.

induced mainly late stage apoptotic cells, which are

comprised of both apoptotic and necrotic cells. Contrary to
the strong apoptotic effect in 1170 cells, DMAPT-induced Fig. (4). Representative Western immunoblots showing concentration-
apoptosis in BEAS-2B cells was much weaker. Moreover, dependent effects of DMAPT on STAT3 and related proteins in 1170
although the apoptosis rate increased significantly in cells and BEAS-2B cells. Cells were treated with different concentrations of
exposed to 15 and 20
M of DMAPT, these cells were DMAPT for 48h, cell lysates prepared and analyzed by Western
predominantly in late stage apoptosis. The percentage of immunblotting as described in the Materials and Methods section. At
cells in early stage apoptosis did not increase significantly at least three independent assays were carried out using cell lysates
any of the concentrations of DMAPT tested (data not shown). prepared on different days. C, vehicle control.
Lung Cancer Chemoprevention with DMAPT Current Cancer Drug Targets, 2014, Vol. 14, No. 1 63

pathways [14, 22-25]. Therefore, we sought to determine

modulation of these pathways by DMAPT in 1170 bronchial
cells and parental BEAS-2B cells. As shown in Fig. 4, in line
with the results of MTT and apoptosis assays, exposure of
1170 cells to a broad concentration range of DMAPT
induced a concentration-dependent suppression of pSTAT3
activation. In BEAS-2B cells, marked suppression of
pSTAT3 was induced only at 15 and 20 M of DMAPT. In
1170 cells, DMAPT also reduced total STAT3 expression at
a concentration of 20 M. Among the down-stream effectors
of STAT3, levels of Mcl-1, cyclin D1 and survivin were
reduced in 1170 cells. Expression of Mcl-1 was decreased at

10 M of DMAPT and a cleaved band was observed at
higher concentrations. Other studies also reported cleavage
of Mcl-1 in cells undergoing apoptosis and this has been
shown to result in loss of the putative BH4 homology
domain that is supposed to be required for the antiapoptotic
activity of all the Bcl-2 family members [26]. The expression
of cyclin D1 was slightly increased at 5 M but decreased at
10 M and completely abolished at 15 and 20 M. DMAPT
reduced the expression of survivin at concentration levels
(15 and 20 M) that dramatically decreased the expression
of pSTAT3, Mcl-1 and cyclin D1.
Since both MAPK-ERK, PI3K/AKT, and NF-
B
signaling play crucial roles in the proliferation and survival
of lung cancer cells, we also assessed the effect of DMAPT
on pERK, pAkt, and IKK-, a key regulator of NF-
B
activation. As shown in Fig. 4, ERK activation was increased
by DMAPT in a concentration-dependent manner in both
BEAS-2B and 1170 cells, but the level of total ERK was not
altered. Expressions of pAkt and IKK- were reduced at the Fig. (5). Representative Western immunoblots showing time-dependent
highest concentration of DMAPT in 1170 cells but not in effects of DMAPT on STAT3 and related proteins in 1170. Cells were
BEAS-2B cells. treated with 10 M of DMAPT for 6, 12, 24, and 48 h, cell lysates
prepared and analyzed by Western immunblotting as described in the
DMAPT modulated expressions of pSTAT3, Mcl-1, Materials and Methods section. At least three independent assays were
cyclin D1 and survivin in 1799 and 1198 cell lines in a carried out using cell lysates prepared on different days. C, vehicle
manner similar to that observed in 1170 cells. Among the control.
transformed cells, DMAPT showed the strongest effects in
1799 cells (data not shown). attenuates the effects of DMAPT (10 M) on pSTAT3,
STAT3-regualted proteins such as Mcl-1, cyclin D1, and
Time-dependent Effects of DMAPT on STAT3 and
survivin and PARP cleavage. As expected, treatment with
STAT3-related Proteins in Bronchial 1170 Cells IL-6 induced over-expression of pSTAT3 without affecting
Exposure of 1170 cells to DMAPT (10 M) for 6, 12, 24 total STAT3 and clearly attenuated the effect of DMAPT
and 48 h, led to a time-dependent suppression of pSTAT3 on pSTAT3 (Fig. 6). These effects were paralleled by
and STAT3-related proteins (Fig. 5). Among the downstream suppression of PARP cleavage in cells exposed to the drug
effectors of STAT3, Mcl-1 was over-expressed and cleaved for 24 and 48 h as compared to cells treated with DMAPT
during the early time points (6 and 12 h), whereas longer only. However, pretreatment with IL-6 did not modulate the
treatment down-regulated the level of the protein. Cyclin D1 effect of DMAPT on Mcl-1, cyclin D1 or survivin.
expression was suppressed at all times points, while the level
of survivin was decreased only after treatment for 48 h. DMAPT Inhibited the DNA Binding Activity of STAT3
Although DMAPT modulated levels of pSTAT3, Mcl-1 and But not that of NFkB in 1170 Cells
cyclin D1, beginning at 6h, PARP cleavage, a marker of Before analyzing the DNA binding activities of STAT3
apoptosis, was observed only at 24 h and 48 h time points. and NFkB, we determined levels of pSTAT3 and p-p65 in
nuclear extracts of DMAPT-treated BEAS-2B and 1170
IL-6 Attenuates DMAPT-induced Suppression of cells. These studies showed a marked reduction of pSTAT3,
pSTAT3 in Bronchial 1170 Cells but not p-p65, in the nuclear fraction of 1170 cells (data not
In order to determine if indeed suppression of pSTA3 by shown), which could suggest suppression by DMAPT of
DMAPT played a role in the drug`s anti-proliferative and STAT3-DNA binding in these cells. To corroborate these
apoptotic effects, we exposed the cells to IL-6, a known results, we assessed STAT3- and NFkB-DNA binding, using
upstream activator of STAT3, and assessed whether IL-6 the TransAM transcription factor assay kit (quantitative
64 Current Cancer Drug Targets, 2014, Vol. 14, No. 1 Song et al.

Fig. (6). IL-6 treatment attenuated the effects of DMAPT on pSTAT3 and PARP cleavage. Bronchial 1170 cells were treated with DMAPT
or/and IL-6 for 6-48 h and expression of the various proteins were determined by Western immunoblotting.

assay) and Odyssey Infrared EMSA kit (qualitative assay), in were given intranasal instillations of DMAPT for 13 weeks
nuclear fractions of PBS- or DMAPT-treated BEAS2B and (Fig. 8A). Weekly mouse body weight measurements
1170 cells. As depicted in Fig. 7A, analysis of nuclear showed that the body weight gain of DMAPT-treated mice,
fractions from 1170 cells treated with DMAPT (10
M), as compared to that of vehicle-treated mice, was reduced by
using TransAM transcription factor assay kit, showed a a maximum of 5 %, indicating that the drug was well-
significant reduction (39%) in STAT3-DNA binding as tolerated (data not shown). In earlier studies, we examined if
compared to the level in PBS-treated control cells. However, long-term intranasal instillation of the chemopreventive
in BEAS-2B cells treated with DMAPT under the same agent diindolylmethane cause undesirable effects to the
conditions, no significant change in STAT3-DNA binding upper/lower respiratory tract [27] and the results revealed
was observed. Also, assessment of the potential modulation neither macroscopic nor microscopic changes associated
of NFkB-DNA binding by DMAPT in nuclear fractions of with intranasal instillation of the agent.
both BEAS2B and 1170 cells did not reveal differences
between PBS-and DMAPT-treated cells (Fig. 7B). Mice treated with NNK and given the vehicle
intranasally had an average of 20.8 ± 8.1 lung tumors/mouse,
Modulation of STAT3-DNA binding by DMAPT was whereas the group treated with NNK and given DMAPT
further corroborated in nuclear fractions of 1170 cells intranasally developed 12.7 ± 4.3 lung tumors/mouse,
analyzed using Odyssey Infrared EMSA kit. Similar to the corresponding to a significant reduction of tumor multiplicity
results observed with the ELISA kit, STAT3-DNA binding by 39% (Table 1). Moreover, classification of NNK-induced
was reduced in a dose-dependent manner, whereas no such tumors according to their sizes indicated that DMAPT
effects were observed in DMAPT-treated BEAS2B cells significantly reduced the multiplicity of bigger tumors. The
(Fig. 7C). number of lung tumors with a diameter of 0.5 - 1 mm was
reduced by 62% in the NNK + DMAPT group, as compared
Effects of Intranasally Administered DMAPT on NNK-
to the number of tumors in mice treated with NNK alone
induced Lung Tumor Multiplicity and Size
(Fig. 8B), whereas multiplicities of smaller tumors (diameter
In order to determine if the in vitro anti-proliferative less than 0.5 mm) were the same in the two groups of mice.
effects of DMAPT will be paralleled by in vivo antitumor On the basis of our extensive experience in mouse lung
effects, mice pretreated with the lung carcinogen NNK tumor bioassay [28], almost all of these lung tumors are
Lung Cancer Chemoprevention with DMAPT Current Cancer Drug Targets, 2014, Vol. 14, No. 1 65

Fig. (7). Effect of DMAPT on DNA binding of STAT3 (A and C) and NFkB/p65 (B) in BEAS-2B and 1170 cells. For STAT3 (A) or
NFkB/p65 (B) DNA binding using ELISA assay, nuclear extracts prepared from BEAS-2B and 1170 cells treated with DMAPT (5, 7.5 or 10

M) for 48 h were processed as described in the Material and Method section. Wild type competitor oligonucleotide (CO) is an unlabelled
oligonucleotide competitor, which inhibits the binding of protein to the probe. Nuclear extracts from HepG2 cells stimulated with IL6 were
used as positive control to check the specificity of the assay. The results are presented as relative percent values compare with control in
BEAS and 1170 cells and represents mean ± SD of two independent experiments with triplicate samples. *P<0.05 vs control sample in each
group (BEAS-2B and 1170). Inhibitory activities of DMAPT on STAT3 DNA binding as determined by EMSA qualitative DNA binding
assay (C). Nuclear extracts from BEAS-2B or 1170 cells treated with DMAPT (5, 7.5 or 10
M) for 48 h were used in the assay as described
in Material and Methods. CO: wild type competitor oligonucleotide.

expected to be at adenoma stage and therefore there was no lung tumor tissues. As shown in Figs. 8C and 8D, consistent
need for histopathological analyses of the tumors. with the results observed in cell line models, DMAPT
significantly reduced the expression of pSTAT3 and Mcl-1
Moreover, we examined modulation by DMAPT of the
in lung tissues from NNK-treated mice. The expression of
expression of pSTAT3 and related proteins in NNK-induced
66 Current Cancer Drug Targets, 2014, Vol. 14, No. 1 Song et al.

Fig. (8). Lung tumor inhibitory effects of DMAPT in A/J mice. A, Experimental design for lung tumor bioassay. B, DMAPT selectively
reduced the multiplicity of larger lung tumors (0.5 – 1 mm). Up on counting of tumors on the surface of the lung, the size of the tumors was
determined and classified into different size categories C, D, Representative Western immunblotting results showing modulation by DMAPT
of STAT3 and related proteins in lung tissues of mice. Lung tissues from vehicle-, NNK- and NNK + DMAPT-treated mice (three
mice/group selected randomly out of 10-14 mice/group used in the tumor bioassay), were individually processed as described in the
Materials and Methods section and the expression of the various proteins was determined by Western immunoblotting. The experiments were
repeated three times. *P < 0.05.

Table 1. Effects of intranasally administered DMAPT on NNK-induced lung tumors in A/J mice.

Group Treatment No. of Mice Body Weight, g Lung Tumor

Incidence (%) Multiplicity Reduction in Tumor p-value*

Multiplicity (%)

1 NNK 14 22.34 ± 0.94 100 20.80 ± 8.07

2 NNK+DMAPT 10 21.14 ± 1.21 100 12.70 ± 4.30 39* 0.02

3 Vehicle control 10 22.2 ± 1.6 20 0.2 ± 0.2

Note: All mice, with the exception of mice in group 3, received NNK (intraperitoneally, at a dose of 50 mg/Kg, once a week, for a total of two doses) in 0.3 ml physiological saline
solution. Beginning one week after the last dose of NNK, mice in group 2 received DMAPT intranasally at a dose of 10 mg/kg mouse, twice a week, until termination of the study at
week 12 (for a period of 24 weeks). Upon termination of the study, tumors on the surface of the lung were counted and tumor multiplicities determined.
*Compared with Group 1.

the other proteins was modulated in a non-significant manner vitro model of lung tumorigenesis consisting of human
or not at all. bronchial cells at different stages of cell transformation, and
an in vivo mouse model of lung tumor. In cell line models,
premalignant cells and malignant cells were more sensitive to
DISCUSSION
the anti-proliferative and apoptotic effects of DMAPT than
In the present work, we assessed the potential lung the parental BEAS-2B cells and these effects were paralleled
cancer chemopreventive activities of DMAPT using an in by modulation of pSTAT3, Mcl-1 and cyclin D1 expression.
Lung Cancer Chemoprevention with DMAPT Current Cancer Drug Targets, 2014, Vol. 14, No. 1 67

Likewise, in mouse models, DMAPT caused a significant cancer. It is primarily activated via phosphorylation of a
decrease in lung tumor number and size and these effects critical tyrosine residue (Tyr 705) that induces STAT3
were paralleled by reduced expression of pSTAT3 and Mcl-1. dimerization through phosphotyrosine-SH2 domain [38].
Activated nuclear STAT3 has been detected in many forms of
Although parthenolide, the principal bioactive component
cancer, including lung cancer [36, 39, 40]. Once dimerized,
of feverfew and the parent compound for DMAPT, possesses STAT3 enters the nucleus and activates a broad array of
significant anticancer effects against many cancer cell
target genes, including the anti-apoptotic proteins Mcl-1 and
lines and has a good safety profile, its bioavailability and
survivin and the pro-growth protein cyclin D1. In our studies
distribution to target tissues is very low, mainly due to poor
with transformed bronchial cells, DMAPT-induced suppression
water solubility [29]. This led to the development of
of pSTAT3 was paralleled by down-regulation of Mcl-1
DMAPT, a water soluble analogue of parthenolide [17,18].
cyclin D1 and survivin and PARP cleavage, indicating
However, although the bioavailability of DMAPT was much that the anti-proliferative and apoptotic effects of DMAPT
better than that of parthenolide, administration of the agent
could be mediated, at least in part, through modulation of
to mice at a dose level of 100 mg/kg achieved only a serum
pSTAT3 and its down-stream effectors. This claim has been
concentration of 25
M and had a half life of 0.63 h [18].
corroborated by the studies with IL-6 in which IL-6 induced
Therefore, we sought to administer DMAPT directly into the
over-expression of pSTAT3 attenuated the effect of DMAPT
lung by intranasal instillation in view of the large surface
on the protein and PARP cleavage at 24 h and 48 h.
area and rich vascular network of the lung which could lead
to a high drug concentration in the lung, a rapid onset of We showed in the present report that DMAPT is a
action, the possibility to avoid drug inactivation resulting promising lung cancer chemopreventive agent as evidenced
from gastrointestinal and hepatic first-pass effects and the by its anti-proliferative effects in transformed bronchial cells
need for reduced dose and frequency of drugs thereby which was further corroborated in a mouse model of lung
improving efficacy and abating dose-limiting side effects [30]. tumorigenesis. These effects were mediated, at least in part,
In a recent study, we showed that intranasal administration of via inhibition of STAT3 signaling pathway. We further
the chemopreventive agent diindolylmethane caused the same showed the benefits of intranasal delivery of lung cancer
antitumor efficacy as a 9-fold higher oral dose of the agent chemopreventive agents since the large surface area and rich
[27], corroborating increased bioavailability of drugs when vascular network of the lung results in high drug
administered directly to the lung. In the present study, although concentration at the target tissue thereby improving efficacy,
we did not determine the pharmacokinetics properties of reducing drug dose and frequency of administration, and
DMAPT in the lung, we showed that intranasal administration abating dose-limiting side effects.
of DMAPT at a reduced dose and frequency (10 mg/kg,
twice a week) led to a significant suppression of NNK- CONCLUSIONS
induced lung tumors.
Overall, we showed in this study that DMAPT is a
Earlier studies on the anticancer activities of DMAPT promising agent for lung cancer chemoprevention in view of
consistently showed that its effects are mediated via its selective suppression of the proliferation and survival of
inhibition of the NF-B pathway [18, 31, 32]. In the present premalignant and malignant bronchial cells, while causing
study, STAT3 and its downstream effectors Mcl-1, and only minimal effects in parental immortalized bronchial
cyclin D1 were found to be the main targets of DMAPT with cells. Induction of differential effects in transformed cells is
little direct effect on expression of NF-B family of proteins crucial for cancer chemopreventive agents as these drugs are
or NF-B binding to DNA. Moreover, analyses of cells given to apparently healthy populations. The effects in cell
pretreated with IL-6, a potent inducer of STAT3 activation, line models were further corroborated in a mouse model of
indicated that the anti-proliferative and apoptotic effects of lung cancer. Moreover, given the consistent findings that
DMAPT in premalignant and malignant bronchial cells are STAT3 has been activated in more than 50% of lung
mediated, at least in part, by targeting the STAT3 pathway. cancers, targeting this protein and its downstream effectors
The concentration of IL-6 used in this study (20, 000 pg/ml) by DMAPT appears to be a promising approach for lung
was 50 times more than the maximum concentration of IL-6 cancer chemoprevention.
measured in the serum of lung cancer patients [33]. This
could be the reason for the failure of DMAPT to completely CONFLICT OF INTEREST
block IL-6-induced STAT3 phosphorylation. Given the close
association between STAT3 and NF-B [34-36], it is unclear The authors confirm that the findings described in this
why DMAPT failed to modulate NF-B-DNA binding and manuscript have no conflict of interest.
the expression of NF-B pathway-related proteins. One
possible reason is that the effect of DMAPT on NF-B and ACKNOWLEDGEMENTS
STAT3 pathways might vary depending on the cell type We express our gratitude to Bob Carlson, Masonic
used. In line with our findings, in one previous study with Cancer Center, University of Minnesota, for his help in the
hepatocellular carcinoma cells, parthenolide, the parent preparation of the manuscript, and Dr. Klein-Szanto, Fox
compound for DMAPT, failed to modify p65 and p50 Chaser Cancer Center, Philadelphia, for generously
activities but inhibited the JAK-STAT3 axis and enhanced providing us the human bronchial cells. This study was
TRAIL-induced apoptosis [37]. funded by Faculty Start-up grants from Masonic Cancer
STAT3 is a transcription factor that is ubiquitously Center and College of Veterinary Medicine, University of
expressed and controls several physiological processes and Minnesota, to FK.
68 Current Cancer Drug Targets, 2014, Vol. 14, No. 1 Song et al.

ABBREVIATIONS [12] Bedoya, L. M.; Abad , M. J.; Bermejo , P. The role of parthenolide
in intracellular signalling processes: review of current knowledge.
AIN = American institute of nutrition Curr. Signal Transd. T. 2008, 3, 82-87.
[13] Hehner, S. P.; Heinrich, M.; Bork, P. M.; Vogt, M.; Ratter, F.;
BEAS-2B = bronchial epithelial cell line 2B Lehmann, V.; Schulze-Osthoff, K.; Dröge, W.; Schmitz, M. L.
Sesquiterpene lactones specifically inhibit activation of NF-
B by
DMAPT = dimethylaminoparthenolide preventing the degradation of IB-
and IB-. J. Biol. Chem.
1998, 273, 1288-1297.
PBS = phosphate buffered saline solution [14] Sobota, R.; Szwed, M.; Kasza, A.; Bugno, M.; Kordula, T.
Parthenolide inhibits activation of signal transducers and activators
EMSA = electrophoretic mobility shift assay of transcription (STATs) induced by cytokines of the IL-6 family.
Biochem. Biophys. Res. Commun. 2000, 267, 329-333.
FACS = fluorescence activated cell sorting [15] Gopal, Y.; Arora, T. S.; Van Dyke, M. W. Parthenolide specifically
Mcl-1 = myeloid cell leukemia-1 depletes histone deacetylase 1 protein and induces cell death
through ataxia telangiectasia mutated. Chem. Biol. Interact. 2007,
MTT = methylthiazoletetrazolium 14, 813-823.
[16] Curry, E. A. 3rd; Murry, D. J.; Yoder, C.; Fife, K.; Armstrong, V.;
NF-
B = Nuclear Factor Kappa beta Nakshatri, H.; O'Connell, M.; Sweeney, C. J. Phase I dose
escalation trial of feverfew with standardized doses of parthenolide
NNK = 4-(methylnitrosamino)-1-(3-pyridyl)-1- in patients with cancer. Invest. New Drugs 2004, 22, 299-305.
butanone [17] Shanmugam, R.; Kusumanchi, P.; Appaiah, H.; Cheng, L.; Crooks,
P.; Neelakantan, S.; Peat, T.; Klaunig, J.; Matthews, W.; Nakshatri,
PARP = poly ADP ribose polymerase H.; Sweeney, C. J. A water soluble parthenolide analog suppresses
in vivo tumor growth of two tobacco-associated cancers, lung and
STAT3 = signal transducer and activator of trans- bladder cancer, by targeting NF-
B and generating reactive oxygen
cription 3 species. Int. J. Cancer 2011, 128, 2481-2494.
[18] Guzman, M.L.; Rossi, R. M.; Neelakantan, S.; Li, X.; Corbett, C.
A.; Hassane, D. C.; Becker, M. W.; Bennett, J. M.; Sullivan, E.;
REFERENCES Lachowicz, J. L.; Vaughan, A.; Sweeney, C. J.; Matthews, W.;
[1] Siegal, R.; Naishadham, D.; Jemal, A. Cancer statistics, 2012. CA Carroll, M.; Liesveld, J. L.; Crooks, P. A.; Jordan, C. T. An orally
Cancer J. Clin. 2012, 62, 10-29. bioavailable parthenolide analog selectively eradicates acute
[2] Thompson, I. M.; Goodman, P. J.; Tangen, C. M.; Lucia, M. S.; myelogenous leukemia stem and progenitor cells. Blood 2007, 110,
Miller, G. J.; Ford, L. G.; Lieber, M. M.; Cespedes, R. D.; Atkins, 4427-4435.
J. N.; Lippman, S. M.; Carlin, S. M.; Ryan, A.; Szczepanek, C. M.; [19] Neelakantan, S.; Nasim, S.; Guzman, M. L.; Jordan, C. T.; Crooks,
Crowley, J. J.; Coltman, C. A. Jr. The influence of finasteride on P. A. Aminoparthenolides as novel anti-leukemic agents: discovery
the development of prostate cancer. N. Engl. J. Med. 2003, 349, of the NF-
B inhibitor, DMAPT (LC-1). Bioorg. Med. Chem. Lett.
215-224. 2009, 19, 4346-4349.
[3] Meyskens, F. L. Jr; McLaren, C. E.; Pelot, D.; Fujikawa-Brooks, [20] Reeves, P.G.; Nielsen, F. H.; Fahey, G. C. Jr. AIN-93 purified diets
S.; Carpenter, P. M.; Hawk, E.; Kelloff, G.; Lawson, M. J.; Kidao, for laboratory rodents: final report of the American Institute of
J.; McCracken, J.; Albers, C. G.; Ahnen, D. J.; Turgeon, D. K.; Nutrition ad hoc writing committee on the reformulation of the
Goldschmid, S.; Lance, P.; Hagedorn, C. H.; Gillen, D. L.; Gerner, AIN-76A rodent diet. J. Nutr. 1993, 123, 1939-1951.
E. W. Difluoromethylornithine plus sulindac for the prevention of [21] Klein-Szanto, A. J.; Iizasa, T.; Momiki, S.; Garcia-Palazzo, I.;
sporadic colorectal adenomas: a randomized placebo-controlled, Caamano, J.; Metcalf, R.; Welsh, J.; Harris, C. C. A tobacco-
double-blind trial. Cancer Prev. Res. 2008, 1, 32-38. specific N-nitrosamine or cigarette smoke condensate causes
[4] Kinsinger, L. S.; Harris, R.; Woolf, S. H.; Sox, H. C.; Lohr, K. N. neoplastic transformation of xenotransplanted human bronchial
Chemoprevention of breast cancer: a summary of the evidence. epithelial cells. Proc. Natl. Acad. Sci. 1992, 89, 6693-6697.
Ann. Intern. Med. 2002, 137, 59-69. [22] Nakabayashi, H.; Shimizu, K. Involvement of Akt/NF-
B pathway
[5] Lotan, R. Retinoids and apoptosis: implications for cancer in antitumor effects of parthenolide on glioblastoma cells in vitro
chemoprevention and therapy. J. Natl. Cancer Inst. 1995, 87, 1655- and in vivo. BMC Cancer 2012, 12, 453.
1657. [23] Hehner, S. P.; Hofmann, T. G.; Dröge, W.; Schmitz, M. L. The
[6] Nishino, H.; Murakosh, M.; Ii, T.; Takemura, M.; Kuchide, M.; antiinflammatory sesquiterpene lactone parthenolide inhibits
Kanazawa, M.;, Mou, X. Y.; Wada, S.; Masuda, M.; Ohsaka, Y.; NF-
B by targeting the IB kinase complex. J. Immunol. 1999,
Yogosawa, S.; Satomi, Y.; Jinno, K. Carotenoids in cancer 163, 5617-5623.
chemoprevention. Cancer Metast. Rev. 2002, 21, 257-264. [24] Di Fiore, R.; Fanale, D.; Drago-Ferrante, R.; Chiaradonna, F.;
[7] Reid, M. E.; Duffield-Lillico, A. J.; Garland, L.; Turnbull, B. W.; Giuliano, M.; De Blasio, A.; Amodeo, V.; Corsini, L. R.; Bazan,
Clark, L. C.; Marshall, J. R. Selenium supplementation and lung V.; Tesoriere, G.; Vento, R.; Russo, A. Genetic and molecular
cancer incidence an update of the nutritional prevention of cancer characterization of the human osteosarcoma 3AB-OS cancer stem
trial. Cancer Epidem. Biomar. 2002, 11, 1285-1291. cell line: A possible model for studying osteosarcoma origin and
[8] Albanes, D.; Heinonen, O. P.; Taylor, P. R.; Virtamo, J.; Edwards, stemness. J. Cell Physiol. 2013, 228, 1189-1201.
B. K.; Rautalahti, M.; Hartman, A. M.; Palmgren, J.; Freedman, L. [25] Skoumal, R.; Tóth, M.; Serpi, R.; Rysä, J.; Leskinen, H.; Ulvila, J.;
S.; Haapakoski, J.; Barrett, M. J.; Pietinen, P.; Malila, N.; Tala, E.; Saiho, T.; Aro, J.; Ruskoahok, H.; Szokodi, I.; Kerkelä, R.
Liippo, K.; Salomaa, E. R.; Tangrea, J. A.; Teppo, L.; Askin, F. B.; Parthenolide inhibits STAT3 signaling and attenuates angiotensin
Taskinen, E.; Erozan, Y.; Greenwald, P.; Huttunen, J. K.
- II-induced left ventricular hypertrophy via modulation of fibroblast
Tocopherol and -carotene supplements and lung cancer incidence activity. J. Mol. Cell. Cardiol. 2011, 50, 634-641.
in the Alpha-Tocopherol, Beta-Carotene Cancer Prevention Study: [26] Herrant, M.; Jacquel, A.; Marchetti, S.; Belhace`ne, N.; Colosetti,
effects of base-line characteristics and study compliance. J.Natl. P.; Luciano, F.; Auberger, P. Cleavage of Mcl-1 by caspases
Cancer Inst. 1996, 88, 1560-1570. impaired its ability to counteract Bim-induced apoptosis. Oncogene
[9] Sebti, S. M.; Adjei, A. A. Farnesyltransferase inhibitors. In: 2004, 23, 7863-7873.
Seminars in oncology, Elsevier, 2004, pp. 28-39. [27] Qian, X.; Song, J. M.; Melkamu, T.; Upadhyaya, P.; Kassie,
[10] Ulrich, C. M.; Bigler, J.; Potter, J. D. Non-steroidal anti- F. Chemoprevention of lung tumorigenesis by intranasally
inflammatory drugs for cancer prevention: promise, perils and administered diindolylmethane in A/J mice. Carcinogenesis 2013,
pharmacogenetics. Nat. Rev. Cancer 2006, 6, 130-140. 34, 841-849.
[11] Franklin, W. A.; Veve, R.; Hirsch, F. R.; Helfrich, B. A.; Bunn, P. [28] Kassie, F.; Matise, I.; Negia, M.; Lahti, D.; Pan, Y.; Scherber, R.;
A. Jr. Epidermal growth factor receptor family in lung cancer and Upadhyaya, P.; Hecht, S. S. Combinations of N-Acetyl-S-(N-2-
premalignancy. In: Seminars in oncology, Elsevier, 2002, pp. 3-14. Phenethylthiocarbamoyl)-L-Cysteine and myo-inositol inhibit
Lung Cancer Chemoprevention with DMAPT Current Cancer Drug Targets, 2014, Vol. 14, No. 1 69

tobacco carcinogen-induced lung adenocarcinoma in mice. Cancer Stat3 maintains constitutive NF-
B activity in tumors. Cancer Cell
Prev Res. 2008, 1, 285-297. 2009, 15, 283-293.
[29] Sweeney, C. J.; Mehrotra, S.; Sadaria, M. R.; Kumar, S.; Shortle, [35] Bollrath, J.; Greten, F. R. IKK/NF-
B and STAT3 pathways:
N. H.; Roman, Y.; Sheridan, C.; Campbell, R. A.; Murry, D. J.; central signalling hubs in inflammation-mediated tumour
Badve, S.; Nakshatri, H. The sesquiterpene lactone parthenolide in promotion and metastasis. EMBO Rep. 2009, 10, 1314-1319.
combination with docetaxel reduces metastasis and improves [36] Grivennikov, S. I.; Karin, M. Dangerous liaisons: STAT3 and NF-
survival in a xenograft model of breast cancer. Mol. Cancer Ther.
B collaboration and crosstalk in cancer. Cytokine Growth Factor
2005, 4, 1004-1012. Rev. 2010, 21, 11-19.
[30] Jones, N. S.; Quraishi, S.; Mason, J. D. The nasal delivery of [37] Carlisi, D.; D'Anneo, A.; Angileri, L.; Lauricella, M.; Emanuele,
systemic drugs. Int. J. Clin. Pract. 1997, 51, 308-312. S.; Santulli, A.; Vento, R.; Tesoriere, G. Parthenolide sensitizes
[31] Shanmugam, R.; Kusumanchi, P.; Cheng, L.; Crooks, P.; hepatocellular carcinoma cells to TRAIL by inducing the
Neelakantan, S.; Matthews, W.; Nakshatri, H.; Sweeney, C. J. A expression of death receptors through inhibition of STAT3
water-soluble parthenolide analogue suppresses in vivo prostate activation. J. Cell. Physiol. 2011, 226,1632-1641.
cancer growth by targeting NF
B and generating reactive oxygen [38] Yoshimura, A.; Naka, T.; Kubo, M. SOCS proteins, cytokine
species. Prostate 2010, 70, 1074-1086. signalling and immune regulation. Nat. Rev. Immunol. 2007, 7,
[32] Estabrook, N.C.; Chin-Sinex, H.; Borgmann, A. J.; Dhaemers, R. 454-465.
M.; Shapiro, R. H.; Gilley, D.; Huda, N.; Crooks, P.; Sweeney, C. [39] Looyenga, B. D.; Hutchings, D.; Cherni, I.; Kingsley, C.; Weiss, G.
J.; Mendonca, M. S. Inhibition of NF-
B and DNA double strand J.; Mackeigan, J. P. STAT3 is activated by JAK2 independent of
break repair by DMAPT sensitizes non small cell lung cancers to key oncogenic driver mutations in non-small cell lung carcinoma.
X-rays. Free Radic. Biol. Med. 2011, 51, 2249-2258. PloS One 2012, 7, e30820.
[33] Yanagawa, H.; Sone, S.; Takahashi, Y.; Haku, T.; Yano, S.; [40] Haura, E. B.; Zheng, Z.; Song, L.; Cantor, A.; Bepler, G. Activated
Shinohara, T.; Ogura, T. Serum levels of interleukin 6 in patients epidermal growth factor receptor–stat-3 signaling promotes tumor
with lung cancer. Br. J. Cancer 1995, 71, 1095-1098. survival in vivo in non–small cell lung cancer. Clin. Cancer Res.
[34] Lee, H.; Herrmann, A.; Deng, J. H.; Kujawski, M.; Niu, G.; Li, Z.; 2005, 11, 8288-8294.
Forman, S.; Jove, R.; Pardoll, D. M.; Yu, H. Persistently activated

Received: July 12, 2013 Revised: October 04, 2013 Accepted: October 24, 2013