Journal of Cancer Therapy, 2010, 1, 197-204
doi:10.4236/jct.2010.14031 Published Online December 2010 (http://www.scirp.org/journal/jct)
197
17-(Allylamino)-17-Demethoxygeldanamycin
Combination with Curcumin Selectively Targets
Mitogen Kinase Pathway in A Human
Neuroblastoma Cell Line
Aftab Taiyab, Usha Kuppa Srinivas, Amere Subbarao Sreedhar
Centre for Cellular and Molecular Biology, Hyderabad, India.
Received June 8th, 2010; revised June 30th, 2010; accepted July 10th, 2010.
ABSTRACT
Pharmacological inhibition of Hsp90 has emerged as a novel anticancer treatment. In this study we have investigated
the effect of Hsp90 inhibitor drug 17AAG combination with curcumin on human neuroblastoma cells. The 17AAG
treatment of cells for 18 h induced G1/S cell cycle arrest associated with cyclin D1 down regulation, and degradation
of Raf-1 and inactivation of Akt. However, 17AAG treatment activated the mitogen kinase, ERK1, and induced the ex-
pression of stress proteins, Hsp70 and p53. The curcumin treatmen t resulted in G2/M cell cycle arrest and activation of
both Raf1 and ERK1 kinases. The drugs in combination induced proteolytic degradation of Raf1 and Akt, and surpassed
curcumin induced G2/M arrest. The combination treatment additionally inactivated MEK, inhibited activation and nu-
clear localization of ERK1, and also inhibited the stress protein induction. EGF stimulation induced re-activation of
mitogen signaling with individual drug treatments but not in combination. This study highlights that 17AAG combina-
tion with curcumin selectively targets mitogen signal transduction mechanism through ERK1 inactivation. In conclu-
sion, our study proposes the beneficial effects of 17AAG combination with curcumin in combating cancer.
Keywords: Hs p90, 17AAG, Curcumin, Neuroblastoma, Combinatorial treatment
1. Introduction
Heat shock protein 9 0 (Hsp90) has shown to be involved
in the conformational maturation and functional stabili-
zation of several oncogenic proteins. Thus, inhibition of
Hsp90 chap erone function usin g 17AAG (17-allylamin o-
17-demethoxygeldanamycin) induces antitumor effects
in tumor models [1,2]. Hsp90 inhibitors either induce
cytostasis and/or apoptosis, therefore treatment of can-
cers with Hsp90 inhibitor drugs is thought to be irre-
versible [3,4]. Curcumin has been shown to elicit cyto-
toxicity in tumor cells throu gh th e activation of apop tosis
[5,6]. Curcumin induced antitumor effects were primar-
ily attributed to its inhibitory role against certain tran-
scription factors [7], and are independent of heat shock
protein inhibition [8].
Cell signaling pathways have been identified as poten-
tial pharmacological targets in antitumor treatments,
therefore protein kinases are considered to be potential
biomarkers [9,10]. Hsp90 stabilizes tyrosine kinases such
as src, Lck, Abl, erb etc., and tyrosine kinase based
growth factor receptors such as IGF (insulin growth fac-
tor), EGF (epidermal growth factor) and PDGF (platelet
derived growth factor). In addition to tyrosine kinases,
Hsp90 also interacts with serine-threonine kinases [11].
The Mitogen activated protein kinase (MAPK) pathways
play important role in cell proliferation and survival and
within the group of MAPKs, oncogenic transformation
has been analyzed primarily in the context of signaling
through Ras-Raf-MEK (MAP kinase kinase) leading to
the activation of ERK (extracellular signal-regulated
kinase) [12]. Cancer is a polygenic disease therefore tar-
geting single kinase or pathway possibly will not account
for effective combating of cancer since these cells have
potential to activate alternate signa ling [13].
In the recent years the combinatorial drug chemistry
has emerged as a powerful tool for anticancer treatments.
17AAG combination with cytoskeletal protein inhibitors

17-(Allylamino)-17-Demethoxygeldanamycin Combination with Curcumin Selectively Targets
Mitogen Kinase Pathway in A Human Neuroblastoma Cell Line
198
such as taxol was reported to be effective against cancer
[14]. Similarly curcumin combination with EGCG (epi-
gallocatechin gallate), cisplatin and doxorubicin were
shown to target pro-survival pathways via NF-kB (nu-
clear factor-kappa B) [15]. There were reports that cur-
cumin combination with piplartine augments the cyto-
toxic effects via ERK and cdk2 (cyclin dependent
kinase-2) inactivation [8], and its combination with vi-
tamin D3 induces differentiation [16]. Considering the
present clinical interest with Hsp90 inhibitors and cur-
cumin in anticancer treatments, we have examined and
evaluated the effect of 17AAG in combination with cur-
cumin in human neuroblastoma tumor cells, and report
that the combination treatment effectively targets mito-
gen signaling through ERK1.
2. Materials and Methods
2.1. Cell Culture and Chemicals
The human neuoroblatoma tumor cells (IMR32) were
maintained in Dulbecco’s Modified Eagles Medium
(DMEM) containing 10% fetal calf serum (FCS), penicil-
lin (100 U/ml), and streptomycin (50 µg/ml) in a humidi-
fied incubator chamber (37C) supplied with 5% CO2. All
the chemicals are procured from Sigma Chemical Com-
pany, USA unless otherwise indicated.
2.2. Drug Treatments
Exponentially growing tumor cells (1x106/ml) in complete
medium were treated with different concentrations of
17AAG (Invitrogen; 0.5 to 10 µM), and curcumin (Sigma
Aldrich; 1 to 25 µM) either alone or in combination. The
EGF at a concentration 50 ng/ml (S igma Aldrich) was used
to confron t drug tre atments. All the treat ments were con tin-
ued with respective time intervals at 37 C bef ore ha rve stin g
for further experiments.
2.3. The Morphology, Viability, and FACS
Analysis
Cells after respective drug treatments were subjected to
morphological examination using a regular phase con-
trast microscope (Nikon, TMS) attached to a 35 mm
camera. Cell viability was determined by trypan blue
exclusion assay before processing cells for further ex-
periments. Control and drugs treated cells were washed
with PBS (phosphate buffered saline), stained with 50
µg/ml propidium iodide (containing 0.1% triton X-100
and 22 µg/ml RNase), and analyzed in a fluorescence
activated cell sorter (FACS Calibur, BD).
2.4．MTT (3-(4,5-Dimethylthiazol-2-yl) -2,
5-diphenyltetrazolium bromide) Assay
Neuroblastoma cells (3x105 cells/ml) were grown on 96
well plates, and the final volume of the medium in each
well was maintained to 100 µl. Cells were treated with
17AAG, curcumin and their combination for 24 h fol-
lowed by treatment with 10 µl MTT (100 mg MTT/ 20
ml DMEM), for 6 h at room temperature with gentle
shaking. The absorbance recorded at 590 nm using
ELISA reader, and the formazan values obtained were
interpr eted such tha t an increase in ab sorbance is ch arac-
teristic to increased cell death.
2.5. Immunofluorescence and Laser Scanning
Confocal Microscopy
Neuroblastoma cells grown on cover slips (Fisher Scien-
tifics, 22x22 mm) after respective drug treatments were
rinsed with PBS, fixed in 3.7% paraformaldehyde for 10
min, and permeabilized with 0.2% Triton X-100 for 10
min. Cells were then washed with PBS, blocked with
PBS-Tween 20 (0.5%; PBS-T) containing 2% BSA for
30 min, and incubated with primary antibody. After be-
ing washed with PBS-T, cells were incubated with cor-
responding FITC-conjugated secondary antibody (Ban-
galore Genie). Cover slips were mounted on slides with
Slow-Fade mounting medium (Molecular Probes) and
analyzed using a laser scanning confocal microscope
(Olympus FV500 microscope).
2.6. SDS-Polyacrylamide Gel Electrophoresis
(SDS-PAGE) and Western Blot Analysis
Cells after respective drug treatments were lysed using
the lysis buffer (20 mM HEPES lysis buffer, pH 7.6 , 10
mM NaCl, 1.5 mM MgCl2, 0.1% Triton X-100, 1 mM
dithiothretol) for 60 min at 4C. Protein concentration
was estimated by Bradford method [17] using bovine
serum albumin (BSA) as standard. Twenty micrograms
of total cell lysate was mixed with Laemmli buffer [18]
containing 100 µl dithiothreitol, boiled for five minutes,
and run on 10% SDS-PAGE. Proteins were transferred
from the gel to nitro-cellulose membrane using a semidry
protein gel transfer apparatus (Amersham Biosciences).
Transfer of proteins was confirmed by Ponceau-S stain-
ing before processing gels for Western blot analysis.
Membrane blocked with 3% BSA followed by incuba-
tion with appropriate primary antibody for 1 h and sub-
sequent incubation with horseradish peroxidase-conjugated
secondary antibody (dilution, 1:3000) for 1 h. The anti-
gen- antibody interaction was visualized using luminol
from BM-Chemiluminescence kit (Roche-Switzerland),
and the protein bands exposed to X--ray film (Kodak).
Antibodies used in the present study, ERK1 (1:1000),
MEK1/2 (1:100 0), Raf 1 (1 :10 00), A k t (1 :800), cyclin D 1
(1:800) from Santa Cruz (USA). The antibodies actin
(1:500), -tubulin (1:500), Hsp70 (1:1000), Hsp27

17-(Allylamino)-17-Demethoxygeldanamycin Combination with Curcumin Selectively Targets
Mitogen Kinase Pathway in A Human Neuroblastoma Cell Line
199
(1:1000), p53 (1:500), and p21 (1:750) are from Stress-
gen, (USA). The dilutions were mentioned after each
respective antibody in closed brackets.
2.7. Statistical Analysis of Data
Data reported as mean ± SD value from the average of
three independent experiments. The control groups were
compared with drug treated ones, and the significance
values were calculated by paired student’s t-test. The P
values represented are *** P < 0.001, ** P < 0.01, and *
P < 0.05”.
3. Results
3.1. 17AAG and Curcumin both Induces
Dose-Dependent Cytotoxicity in Human
Neuroblastoma
Different concentrations of 17AAG were examined
against neuroblastoma cells for 18 h, and the cytotoxicity
was measured by MTT assay. We observed a concentra-
tion dependent cytotoxicity reaching 62% at 10 µM con-
centrations (Figure 1(a)). Similarly, curcumin treatment
for 24 h showed cytotoxicity only at above 20 µM con-
centration. Notably 10 µM 17AAG showed >50 % cell
death, and maximum cytotoxicity (100%) observed with
curcumin was at 25 µM concentration (Figure 1(b)).
Considering minimal cell death induced by these drugs
than reported at low concentrations, we have used com-
bination treatment of 17AAG (2 µM) with curcumin (15
µM). The combination treatment howev er not resulted in
any significant cell death in 24 h treatment (Figure 1(c)).
Therefore we analyzed for potential effect of individual
drugs on cell cycle, and observed 17AAG induced G1/S
and curcumin induced G2/M arrest. Interestingly, in the
Figure 1. 17AAG, curcumin, and their combination on drug
induced cytotoxicity. (Figure 1(a), 1(b), and 1(c)). Control
values are normalized with treatment in Figure 1(a). Cell
cycle analysis of drug treated neuroblastoma shows differ-
ential cell cycle arrest (Figu r e 1 ( d )).
combination treatment, 17AAG surpassed curcumin in-
duced G2/M arrest and thus arrested cells in G1/S phase
of cell cycle (Figure 1(d)).
3.2. 17AAG and Curcumin Combination
Selectively Targets Mitogen Signaling
Pathway
Anti-Hsp90 drugs dissociate Hsp90 from Hsp90-client
protein interaction that induces destabilization of client
proteins [11]. However, curcumin induced proteolytic
degradation of oncogenic kinases was not reported [19].
Therefore, the effect of individual drugs and their com-
bination was tested against activation and degradation of
various serine-threonine kinases such as Raf1, Akt, MEK
and ERK kinases. We observed that while curcumin
treatment was inducing Raf1 activation, 17AAG treat-
ment resulted in its proteolytic degradation. The basal
expression of Akt w a s though not affected by these drugs,
17AAG alone and in combination with curcumin inhib-
ited Akt activation. While individual drug treatments
increased ERK1 phosphorylation, the combination
treatment resulted in significant inhibition of its activa-
tion, and additionally induced its degradation. The com-
bination treatment further resulted in induced degrada-
tion of MEK (Figure 2).
3.3. 17AAG Treatment Surpasses Curcumin
Induced Cycle Arrest
Cell cycle machinery controls cell proliferation, and
cancer is a disease of inappropriate cell proliferation. In
Figure 2. Drugs induced differential signaling response.
Note only 17AAG and its combination with curcumin in-
duced inactivation of Akt followed by inactivation of MEK
and ERK. The densitometric plots shows fold decrease.
Actin was used as loading control.

17-(Allylamino)-17-Demethoxygeldanamycin Combination with Curcumin Selectively Targets
Mitogen Kinase Pathway in A Human Neuroblastoma Cell Line
200
addition to kinase specific inhibitors that also inhibit cell
division, irreversible cell cycle inhibition alone is
thought to be effective in successful combating of cancer
[20]. The cell cycle inhibition usually correlates with
increased p21 and decreased cyclin D1 expressions,
therefore we analyzed for the expression of these mole-
cules by immunoblot analysis. We observed a correlation
of our cell cycle study with induced expression p21 and
decreased levels of cyclin D1 (Figure 3).
3.4. Curcumin Combination Inhibits the Stress
Response Induced by 17AAG
Usually cell cycle arrest proceeds to either apoptosis or
differentiation [21]. From the analysis of neuronal dif-
ferentiation markers such as Map2, Gad67, heavy neuro-
filament we found that these drugs do not induce differ-
entiation (data not presented), nevertheless, accumulation
of cells in G1/S phase of cell cycle suggested activation
of stress response. In contrast to curcumin, 17AAG
treatment induced Hsp70 and p53 protein expression,
whereas curcumin and 17AAG treatments have inde-
pendently induced Hsp27 expression, however, the com-
bination treatment has resulted in the inhibition of Hsp70,
p53 and Hsp27 induction. A diminutive decrease in
Hsp90 expression was observed upon 17AAG and its
combination with curcumin treatments (Figure 4).
3.5. Effect of 17AAG and Curcumin on ERK1
Signaling
MAPK pathway plays an important role in signal trans-
duction in response to a wide variety of extracellular
signals [22]. Since combination drug treatment resulted
in the inactivation of ERK1 despite of its stable activa
Figure 3. Drugs induced effects on cell cycle proteins. Note
a significant decrease in cyclin D1 levels, and a gradual
increase in p21 levels after 17AAG treatment and its en-
hancement in combination treatment with curcumin. The
densitometric plots shows fold decrease in the expression of
signaling molecule. Actin immunoblot used as loading con-
trol.
Figure 4. Drugs effect on stress proteins. Note 17AAG treat-
ment induced expression of Hsp70, p53 and both curcumin
and 17AAG induced Hsp27 levels. Also note that inhibition of
stress response in the combination treatment. The densi-
tometric plots shows fold decrease in the expression of sig-
naling molecule. Actin immunoblot used as loading control.
tion in individual drug treatments (Figure 2), we exam-
ined for ERK1 cellular localization by immunofluores-
cence analysis. From the immunoflourescence study in
control cells, phosphorylated ERK1 localization was ob-
served both in peri-nuclear and cytoplasmic accretion;
however, curcumin and 17AAG treatments induced its
nuclear translocation. In the combination treatment, we
observed both inactivation as well as decreased nuclear
Figure 5. Drugs induced re-localozation of activated ERK1
by confocal scanning microscopy. Rows represents control,
curcumin, 17AAG, and 17AAG in combination with curcu-
min, and the columns represents ERK1 (green), nucleus
staining with DAPI (blue), and merge (green + blue). Note
ERK1 nuclear localization in curcumin and 17AAG treat-
ments compared to control, and inability in nuclear localiza-
tion treament.

17-(Allylamino)-17-Demethoxygeldanamycin Combination with Curcumin Selectively Targets
Mitogen Kinase Pathway in A Human Neuroblastoma Cell Line
201
localization of ERK1 (Figure 5).
3.6. Effect of Epidermal Growth Factor in the
Reversal of Mitogen Signal Inactivation
The membrane receptors, including EGF, transduce sig-
nals by activating the MAPK family of proteins; there-
fore tumor cells take advantage of these kinases for con-
stitutive activation of survival signals. In the present
study, to examin e whether our combination is selectively
targeting the mitogen signaling pathway, we evaluated
EGF effect in 17AAG and curcumin combination drug
pre-treatment (EGF treatment followed by combination
drugs), co-treatment (EGF treatment along with combi-
nation of drugs) and post-treatments (EGF treatment af-
ter the combination drug treatment). The EGF treatment
induced reversal of cytotoxicity in the combination
treatments significantly, however, was not found to be
effective with 17AAG treatment alone. Among the pre-,
co-, and post- EGF treatments, the co- and
post-treatments showed decreased cell recovery com-
pared to the pre-treatment (Figure 6). The improved
growth in EGF pre-treatment could be related to en-
hanced proliferative signaling prior to drugs treatment,
whereas co- and post-treatments suggests absence or
lowered proliferation rate due to combination of drugs.
These findings invoked existence of possible signal
transduction dependent regulatory mechanism operated
by EGF in the reversal of 17AAG effect however on ly in
the combination treatment. In support, the immunoblot
analysis of EGF treated cells showed restrained Raf1,
Akt and MEK activities but resulted in enhanced prote-
olytic degradation of ERK1 (Figure 7).
4. Discussion
Among several anticancer drugs that are under clinical
evaluation, Hsp90 inhibitor drug 17AAG [23-24] and an-
tioxidant and anti-inflammatory drug curcumin [6] are
attributed for their selectivity and specificity against a
large variety of cancer cells. Considering the growing in-
terest and beneficial effects of combinatorial drug treat-
ments in treating cancer, we have examined for combina-
torial effects of 17AAG and curcumin against human
neuroblastoma tumor cells. From the present study, we
report that the combination drug treatment selectively tar-
gets the MAP kinase signal transducti on pathway.
Several reports of 17AAG combination with ox-
aliplatin, flurouracil [25], carboplatin [26], paclitaxel
[27], rapamycin [28], trastuzumab and tanespinycin [29],
histone deacetylease [30] etc., suggests that 17AAG
combination treatments not only enhance drug specific
effects, but exhibit synergistic effects however not
though MAP kinases. In a classical mitogen activated
Figure 6. Epidermal growth factor (EGF) treatment on cell
recovery and mitogen signal activation. Compared to
17AAG treatment its combination with curcumin shows
reversal of phenotype. Note that only post-treatment of cells
with EGF shows maximum survival compared to co- and
pre-treatments.
Figure 7. Drugs induced differential signaling response
after EGF recovery. Note only 17AAG and its combination
with curcumin induced inactivation of Akt, MEK and ERK.
The densitometric plots shows fold decrease. Tubulin used
as loading control.
signaling pathway MEK binds to ERK and this coordi-
nated binding is essential for the activation of ERK. De-
regulated signal transduction mechanism is one of the
hall marks of cancers [31,32]. From our studies it is evi-
dent that Raf1 activation by curcumin or inactivation by
17AAG has no or little effect on MEK and ERK signal-
ing, therefore, the Akt or Raf1 activation appears to be
independent of do wnstream effector s of MAPK pathway.
Usually only one form of dual phopshorylated ERK ex-
ists in the cell with greater specificity, which translocates
to the nucleus and activates certain transcription al factors.
In agreement with curcumin induced ERK and JNK

17-(Allylamino)-17-Demethoxygeldanamycin Combination with Curcumin Selectively Targets
Mitogen Kinase Pathway in A Human Neuroblastoma Cell Line
202
pathways [33], we report ERK1 activation and its nuclear
localization with 17AAG and curcumin, however, the
combination has significantly inhibited its activation and
nuclear translocation .
Despite the fact that curcumin alone can activate stress
response in experimental models [34,35], in our system
curcumin did not induce stress response. Curcumin on
the other hand had inhibited stress response induced by
17AAG. Similar results were obtained with different cell
types such as rat histiocytoma (BC-8 ), rat hepatoma cells
(CRL-1600), human T-lymphocyte cells (Jurkat), mouse
embryonal carcinoma cells (PCC4). Induced stress pro-
teins are known to hamper cancer treatments, therefore,
drugs that induce cytotoxic effects without inducing the
stress response are considered to be effective anticancer
agents [36]. Only a decrease in BCl2 expression in asso-
ciation with increased stress proteins further suggests
activation of only the stress response, but not apoptotic
response. Treatment of tumor cells with ROS (reactive
oxygen species) scavenger, NAC (N-acetyl cysteine),
ascorbic acid, vitamin K etc., though decreased ROS
levels, they did not show any effect on combination
treatment induced mitogen signal targeting (data not
shown). Contrary to curcumin induced p21 and degrada-
tion of cyclin D1 [5,37], we did not find curcumin tar-
geting of these two molecule. However, once again it is
only the combination treatment that had resulted in the
degradation of cyclin D1, which is otherwise found de-
regulated in majority of cancers [38].
The EGF and EGF-receptor signaling stimulates
growth of cells in culture without affecting DNA synthe-
sis [39]. Neuroblastoma tumors are known to have in-
duced expression of EGF family of receptors [40]. Ob-
serving a MAPK dependent targeting in our combinato-
rial drug treatment with 17AAG and diferuloylmethane,
we have examined for EGF effect on drug treated cells
and found that EGF stimulation though resulted in the
restitution of normal division potential, selective target-
ing of ERK signaling was established.
We present that 17AAG combination with curcumin
potentiated 17AAG effect and add ition ally targ eted MAP
kinases. Combination treatment further inhibited 17AAG
induced stress response. Our results indicate that 17AAG
may be a promising candidate to use in combinatorial
treatments with curcumin to combat cancer where mito-
gen kinase expression is challenging the anticancer
treatment.
5. Acknowledgements
Authors thank Ms. Nandini for confocal microscopy.
Department of Biotechnology and Department of Sci-
ence and Technology, Ministry of Human Resource De-
velopment, Government of India supported the work in
author’s laboratory.
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