Int. J.
Cancer: 122, 267–273 (2008)
' 2007 Wiley-Liss, Inc.
FAST TRACK
Curcumin enhances the effects of 5-fluorouracil and oxaliplatin in mediating
growth inhibition of colon cancer cells by modulating EGFR and IGF-1R
Bhaumik B. Patel1,2, Radha Sengupta1, Sadia Qazi1, Hetal Vachhani3, Yingjie Yu1,
Arun K. Rishi2 and Adhip P.N. Majumdar1,2,4*
1
John D. Dingell VA Medical Center, Wayne State University, Detroit, MI
2
Department of Internal Medicine, Wayne State University, Detroit, MI
3
Henry Ford Hospital, Detroit, MI
4
Karmanos Cancer Institute, Wayne State University, Detroit, MI
Curcumin (diferuloylmethane), which has been shown to inhibit
growth of transformed cells, has no discernible toxicity and
achieves high levels in colonic mucosa. 5-fluorouracil (5-FU) or 5-
FU plus oxaliplatin (FOLFOX) remains the backbone of colorectal
cancer chemotherapeutics, but with limited success. The present
investigation was, therefore, undertaken to examine whether
curcumin in combination with conventional chemotherapeutic
agent(s)/regimen will be a superior therapeutic strategy for color-
ectal cancer. Indeed, results of our in vitro studies demonstrated
that curcumin together with FOLFOX produced a significantly
greater inhibition (p < 0.01) of growth and stimulated apoptosis
(p < 0.001) of colon cancer HCT-116 and HT-29 cells than that
caused by curcumin, 5-FU, curcumin 1 5-FU or FOLFOX. These
changes were associated with decreased expression and activation
(tyrosine phosphorylation) of EGFR, HER-2, HER-3 (72–100%)
and IGF-1R (67%) as well as their downstream effectors such as
Akt and cycloxygenase-2 (51–97%). Furthermore, while these
agents produced a 2–3-fold increase in the expression of IGF-bind-
ing protein-3 (IGFBP-3), curcumin together with FOLFOX
caused a 5-fold increase in the same, when compared to controls.
This in turn led to increased sequestration of IGF by IGFBP-3
rendering IGF-1 unavailable for binding to and activation of IGF-
1R. We conclude that the superior effects of the combination ther-
apy of curcumin and FOLFOX are due to attenuation of EGFRs
and IGF-1R signaling pathways. We also suggest that inclusion of
curcumin to the conventional chemotherapeutic agent(s)/regimen
could be an effective therapeutic strategy for colorectal cancer.
' 2007 Wiley-Liss, Inc.
Key words: colorectal cancer; EGFR; IGF-1R; curcumin; chemo-
therapy; IGFBP3
There will be an estimated 148,610 new cases and 55,170
deaths due to colorectal caner (CRC) in 2006 in the USA.1 CRC is
estimated to be the second and third leading cause of cancer-
related deaths in men and women, respectively, in 2006.1 Surgery
and subsequent chemotherapy can cure over 75% colon cancer
patients, but more than 30% of these patients develop new neo-
plastic polyps, and 10% progress to frank second malignancy.2–4
The risk of second malignancy is higher for microsatellite instable
tumor (MSI).5 Metastatic colorectal cancer has poor a prognosis
with 5-year survival of less than 10%.6 As a result of great efforts
are being spent on improving chemotherapeutic interventions for
metastatic colon cancer, and the median survival has improved to
over 20 months of this group of patients.7 However, this comes at
a cost of additional toxicities, some of which are even fatal.7 The
validation of a nontoxic agent that could improve upon the current
chemotherapeutic regimen would therefore be highly desirable.
Accumulating evidence suggests that the development and
progression of many malignancies, including colorectal cancer,
are associated with constitutive activation of multiple signaling
pathways that promote proliferation, inhibit apoptosis and induce
metastasis.8 A large body of evidence suggests that EGF-receptor
(EGFR) and/or its family members, specifically ErbB-2/HER-2
and ErbB-3/HER-3 [collectively referred to as EGFRs], play a
crucial role in regulating several pathways that affect tumor cell
survival, angiogenesis, motility and invasiveness.9–11 Recent data
Publication of the International Union Against Cancer
have also implicated IGF/IGF-1R system in the development and
progression of colorectal cancer.12,13 Therefore, agent(s) that
would target EGFRs and IGF-1R are likely to affect multiple
aspects of tumor progression.
Curcumin (diferuloylmethane), the major active ingredient of
turmeric (curcuma longa) with no discernable toxicity, has been
shown to inhibit the growth of transformed cells and colon carci-
nogenesis at the initiation, promotion and progression stages in
carcinogen-induced rodent models.14–18 Curcumin has also been
shown to prevent the development of adenomas in the intestinal
tract of Min1/2 mice, a model of human familial adenomatous
polyposis.19 In a Phase I clinical trial, curcumin has been found to
be effective in inhibiting the growth of a variety of tumors.20
In the current in vitro study, we have examined the effects of
curcumin in combination with most widely used chemotherapeutic
regimen (FOLFOX) in causing growth inhibition of colon cancer
cells. In this communication, we report that the combination of
curcumin and FOLFOX caused a greater growth inhibition of co-
lon cancer cells than that caused by curcumin, 5-FU, curcumin 1
5-FU, curcumin 1 oxaliplatin or FOLFOX alone in 2 different
colon cancer cells (HCT-116 and HT-29) with distinctive genotypic
differences. We also have examined the possible mechanism of
the synergism focusing on the regulation of surface growth factor
pathways, specifically EGFRs and IGF-1R, which are considered
to play important role in the progression of colorectal cancer. The
current study alleviates some concerns raised earlier about possi-
ble antagonism between curcumin and some chemotherapeutic
agents in a breast cancer model and proposes the use of curcumin-
based combination as an effective therapy for colorectal cancer.21
Material and methods
Cell lines and cell cultures
Human colon cancer HCT-116 and HT-29 cells were obtained
from American Type Culture Collection (ATCC, Rockville, MD).
Cells were maintained in tissue culture flasks in a humidified incu-
bator at 37°C in an atmosphere of 95% air and 5% CO2. Medium
was supplemented with 10% FBS and 1% antibiotic/antimycotic.
Medium was changed 3 times a week and cells were passed using
trypsin/EDTA.
Growth inhibition assay
Inhibition of cell growth in response to curcumin and/or
recombinant ERRP was assessed by 3-(4,5-dimethylthiazol-2yl)-
Grant sponsor: NIH/NIA; Grant number: AG 014343; Grant sponsor:
Department of Veterans Affairs.
*Correspondence to: John D. Dingell VA Medical Center, 4646 John
R; Room: B-4238, Detroit, MI 48201. Fax: 1313-576-1112.
E-mail: a.majumdar@wayne.edu
Received 19 April 2007; Accepted after revision 30 July 2007
DOI 10.1002/ijc.23097
Published online 4 October 2007 in Wiley InterScience (www.interscience.
wiley.com).
268 PATEL ET AL.
2,5-diphenyltetrazolium bromide (MTT) assay as described previ-
ously.22 Briefly, cells were dispersed by trypsin-EDTA treatment
and 2.5 3 104 cells/ml resuspended in DMEM containing 10% of
FBS and seeded into 96-well culture plates with six replicates.
After 24 hr of plating, incubation was continued for another 48 hr
in absence (control) or presence of different testing agents as
described in the legends to the figures. At the end of the 48-hr
incubation period, the reaction was terminated by adding 20 ll of
5 mg/ml stock of MTT to each well. The reaction was allowed to
proceed for 3–4 hr at 37°C. The culture medium was then
removed. The formazan crystals were then dissolved by adding
0.1 ml of dimethyl sulfoxide (DMSO). The intensity of the color
developed, which is the reflection of number of live cells, was
measured at a wavelength of 570 nm. All values were compared
to the corresponding controls. All assays were performed with 6
replicates.
Assessment of apoptosis
Approximately 1 3 105 cells/well were plated in DMEM/10%
FBS. After 24 hr of plating, the medium was changed to contain
2.5% FBS to minimize the contribution of serum-derived growth
factors, and subsequently treated the same way as described above
for growth inhibition study. At the end of the incubation period,
the cells were lysed, and the levels of apoptosis were determined
using the Cell Death Detection ELISAPLUS kit from Roche Diag-
nostics GmbH (Penzberg, Germany), which measures the cyto-
plasmic histone-associated-DNA-fragments (mono- and oligonu-
cleosomes).
Western-blot analysis
Western-blot analysis was performed essentially according to
our standard protocol.22 Briefly, the cells were solubilized in lysis
buffer [50 mM Tris; 100 mM NaCl; 2.5 mM EDTA; 1% Triton
X-100; 1% Nonidet P-40; 2.5 mM Na3VO4; 25 lg/ml aprotinin;
25 lg/ml leupeptin; 25 lg/ml pepstatin A; and 1 mM phenylmeth-
ylsulfonyl fluoride (PMSF)]. Following clarification at 10,000g for
15 min, the supernatant was used for Western-blot analysis. In all
analyses, protein concentration, determined by the Bio-Rad Pro-
tein Assay kit (Bio-Rad, Hercules, CA), was standardized among
the samples. Aliquots of cell lysates containing 50 lg of protein
were separated by sodium dodecyl sulfate-polyacrylamide gel
electrophoresis (SDS-PAGE). Following electrophoresis, proteins
were transferred electrophoretically onto supported nitrocellulose
membranes (Osmonics, Gloucester, MA). Membranes were incu-
bated for 1 hr at room temperature with blocking buffer, TBS-T
(20 mM Tris, pH 7.6, 100 nM NaCl, 0.1% Tween-20) and
5% nonfat dry milk with gentle agitation. After washing the
membranes with TBS-T, they were incubated overnight at 4°C in
TBS-T buffer containing 5% milk and with one of the following
antibodies (1:1,000 dilution): phospho-EGFR (Tyr1173), phospho-
ErbB-2/HER-2 (Tyr1121), phospho-ErbB-3/HER-3 (Tyr1289), IGF-
1R, IGFBP-3, Akt (Ser473) or COX-2. The membranes were
washed 3 times with TBS-T, and subsequently incubated with
appropriate secondary antibodies (1:5,000 dilutions) in TBS-T
containing 5% milk for 2 hr at room temperature with gentle agita-
tion. The membranes were washed again with TBS-T, and the pro-
tein bands were visualized by enhanced chemiluminescence
(ECL) detection system (Amersham). The membranes containing
the electrophoresed proteins were exposed to X-Omat film, and
the signals were quantitated by densitometry using Image Quant
image analysis system (Storm Optical Scanner, Molecular Dyna-
mics, Sunnyvale, CA). Membranes were stripped (23 for 15 min at
55°C) in stripping buffer containing 100 mM 2-mercaptoethanol,
2% sodium dodecyl sulfate, 62.5 mM Tris-HCl pH 6.7. The mem-
branes were then reprobed for the levels of total (nonphosphory-
lated) EGFR, ErbB-2, ErbB-3, Akt or b-actin using corresponding
antibodies. All Western-blots were performed at least 3 times for
each experiment.
In experiments where proteins were immunoprecipitated, cell
lysates containing 1 mg protein were incubated for 24 hr at 4°C
FIGURE 1 – Dose-dependant inhibition of growth (MTT assay) of
colon cancer HCT-116 and HT-29 cells in response to curcumin. Cells
were incubated for 48 hr in the absence (control) or presence of curcu-
min.
under constant stirring with appropriate antibodies and protein-G-
Sepharose beads. The beads were initially washed twice with TT
buffer (50 mM Tris, pH 7.6, 0.15 M NaCl, 0.5% Tween 20) and
suspended in TTA buffer (TT buffer 1 0.1% BSA). The incuba-
tion was conducted in a total volume of 0.7 ml TTA buffer that
contained 5 ll antibody and 60 ll of the protein-G Sepharose
beads. Following incubation, the immunoprecipitates were washed
6 times with TT buffer, and subjected to Western-blot analysis as
described above. In the current investigation, the immuoprecipi-
tates containing IGFBP-3 were subjected Western-blot analysis
with IGF-1.
All immunoblots were scanned by HP Precision Pro 3.13 (Hew-
lett–Packard, Packard, Palo Alto, CA). Densitometric measure-
ments of the scanned bands were performed using the digitized
scientific software program UN-SCNAT. Data were normalized to
b-actin.
Reporter gene assay
Cells were transfected with 0.8 lg of pRE260 (which contains
260 nt of EGFR promoter) plasmid and 0.2 lg of internal control
pRSVZ plasmid. After the treatment, the cells were lysed in lysis
buffer (25 mM glycyl glycine, 15 mM MgSO4, 4 mM EGTA, 1%
Triton X-100, and 0.1 mM DTT) and then clarified by centrifuga-
tion at 10,000g for 5 min. The supernatant was used for luciferase
assay. Luciferase activities were measured in a luminometer
(AutoLumat Plus, Berthold Tech). Transfection efficiency was
normalized to galactosidase activity. Values are means 6 SE.
Statistical analysis
Unless otherwise stated, data are expressed as mean 6 SEM.
Where applicable, the results were analyzed using ANOVA fol-
lowed by Fischer’s protected least significant differences or
Scheffe’s test. A p value of <0.05 was designated as the level of
significance.
Results
Dose response study was performed with curcumin in both
HCT-116 and HT-29 colon cancer cells. Curcumin inhibited
growth of both cell lines in a dose-dependant manner, revealing a
close to 90% inhibition at 80 lM concentration (Fig. 1). A 50–
60% growth inhibition was observed in both cell lines at a dose of
10 lM, which was chosen in all subsequent studies (Fig. 1). Stud-
ies were also performed to examine the effect of 5-FU on growth
of colon cancer cells. Like curcumin, 5-FU also inhibited growth
of colon cancer HCT-116 cells (data not shown). A marked 65%
 CURCUMIN AND CHEMOTHERAPEUTIC AGENTS IN COLON CANCER 269

FIGURE 2 – Cell growth inhibition (MTT assay) at 48 hr in (a) HCT-116 and (b) HT-29 cells with curcumin (10 lM), 5-FU (0.2 mM), FOL-
FOX [5-FU (0.2 mM) and oxaliplatin (5 lM)], 5-FU 1 curcumin or FOLFOX 1 curcumin. Values represent the mean 6 SEM of 4–5 observa-
tions. Asterisk (*) sign indicates statistically significant values compared to control (p < 0.01); Plus (1) sign indicates statistically significant
values compared to 5-FU 1 curcumin or FOLFOX (p < 0.01).

FIGURE 3 – Apoptosis induction (ELISA assay) at 48 hr in (a) HCT-116 and (b) HT-29 cells with curcumin (10 lM), 5-FU (0.2 mM), FOL-
FOX [5-FU (0.2 mM) and oxaliplatin (5 lM)], 5-FU 1 curcumin or FOLFOX 1 curcumin. Values represent the mean 6 SEM of 4–5 observa-
tions. Asterisk (*) sign indicates statistically significant values compared to control (p < 0.01); Plus (1) sign indicates statistically significant
values compared to 5-FU 1 curcumin or FOLFOX (p < 0.01).

inhibition was achieved with a dose of 0.1 mM 5-FU with no fur-
ther significant inhibition occurring with concentrations up to 0.8
mM (data not shown). 5-FU at concentrations of 0.2 mM was used
in all subsequent experiments. We did not evaluate the growth in-
hibitory properties of oxaliplatin as a single agent, since it is not
effective when used alone in the treatment of colorectal cancer. In
the current investigation, we used 5 lM oxaliplatin, a dose that
has shown to be effective in vitro.23
The next set of experiments was performed to determine
whether and to what extent curcumin when given together with
the chemotherapeutic agents/regimens would affect colon cancer
cell growth. We observed that although curcumin, 5-FU, curcumin
1 5-FU, FOLFOX (5-FU1Oxaliplatin) were all effective in sig-
nificantly inhibiting the growth of HCT-116 or HT-29 cells by
33–60%, curcumin together with FOLFOX caused even further in-
hibition of growth (68–73%), when compared with the controls
(Fig. 2).
We also observed that combination of curcumin and FOLFOX
caused a marked induction of apoptosis of HCT-116 and HT-29
cells (Fig. 3). HCT-116 cells, treated with curcumin and FOL-
FOX, showed a robust (>10-fold) increase in apoptosis compared
to vehicle-treated cells. The increased apoptosis observed with 3
270 PATEL ET AL.

FIGURE 5 – Effect of curcumin, 5-FU, FOLFOX, 5-FU 1 curcumin

or FOLFOX 1 curcumin on activated and total AKT as well as COX-
2 expression in HCT-116 cells. The cells were treated with respective
FIGURE 4 – Inhibition of EGFRs and IGF-1R expression and activa- agents and combinations for 48 hr before harvesting the protein. The
tion (tyrosine phophorylation) in HCT-116 cells in response to curcu- numbers underneath each band represent percent of representative
min (10 lM), 5-FU (0.2 mM), FOLFOX [5-FU (0.2 mM) and oxalipla- control. The experiment was repeated at least 3 times.
tin (5 lM)], 5-FU 1 curcumin or FOLFOX 1 curcumin. The cells were
treated with respective agents/regimen for 48 hr before harvesting the
protein. The numbers underneath each band represent percent of repre-
sentative control. The experiment was repeated at least 3 times.

drug combination was significantly greater (p < 0.01) than that

observed with curcumin, 5-FU, FOLFOX or curcumin 1 5-FU
(Fig. 3a). A similar trend was observed with HT-29 cells (Fig. 3b).
However, the magnitude of apoptosis induction was modest (3.7-
fold for curcumin and FOLFOX) compared to that observed in
HCT-116 cells. Taken together, the results suggest that curcumin
may act synergistically with the chemotherapeutic regimen of
colon cancer. In addition, the effect is not cell-specific, since it
was observed in both HCT-116 and HT-29 cells.
Although curcumin either alone or in combination with che-
motherapeutic agent/regimen inhibited colon cancer cell growth,
the precise regulatory mechanisms are poorly understood. Ear-
lier, we reported that the marked inhibition of growth of colon
cancer cells in vitro in response to the combinatorial treatment of
curcumin and ERRP, a pan-erbB inhibitor, was associated with
the attenuation of activation of EGFR and IGF-1R signaling.22
Since the current combinatorial treatment of curcumin with FOL-
FOX also produced a greater effect on cell growth than that
caused by either agent/regimen alone, we speculated that a simi-
lar mechanism might be responsible. Therefore, we examined the
constitutive levels of total and the activated (tyrosine phospho-
rylation) forms of EGFR, HER-2 and HER-3 as well as the acti-
vation of IGF-1R in response to these agents. Curcumin by itself
inhibited the activated forms of all EGFRs, but only downregu-
lated total EGFR. On the other hand, 5-FU and FOLFOX caused
downregulation as well as activation of EGFRs. Curcumin when
combined with FOLFOX inhibited both expression and activa-
tion of EGFR, HER-2 and HER-3 in HCT-116 cells to a much
greater extent (72–100%) than that caused by each agent/regimen
alone (Fig. 4).
Like EGFRs, IGF-1R responded in a similar manner with levels
of activated (phosphorylated) IGF-1R being decreased to a greater
extent (67%) in HCT-116 cells in response to the combinatorial
treatment of curcumin and FOLFOX than each agent/regimen
alone (Fig. 4).
We next sought to determine the extent to which the down-
stream events of EGFRs and IGF-1R signaling are affected by cur-
cumin or the chemotherapeutic agents/regimen, alone or in combi-
nation. We examined the levels of total and phosphorylated (acti-
vated) forms of Akt as well as the expression of COX-2, both of
which are critical mediators of cell growth. Consistent with attenua-
FIGURE 6 – Changes in EGFR promoter luciferase activity in HCT-
116 cells in the absence (control) or presence of curcumin, FOLFOX
or FOLFOX 1 curcumin following transfection of EGFR promoter-
luciferase construct.
tion of EGFRs and IGF-1R, curcumin together with FOLFOX
inhibited the expression and activation of AKT (51–73%) and
COX-2 (93%) in HCT-116 cells to a much greater extent than that
caused by curcumin, curcumin15-FU or FOLFOX (Fig. 5). The
above observations suggest that synergistic inhibition growth with
curcumin and FOLFOX combination may be due to decreased
activation of EGFRs and IGF-1R and their subsequent signaling.
To elucidate the mechanism by which chemotherapeutic agents
and curcumin downregulated EGFR, we examined the effect of
these agents on activation of EGFR promoter. We transfected
HCT-116 cells with rat EGFR plasmid construct containing lucif-
erase reporter gene and assayed for luciferase activity 24 hr later.
As shown in Figure 6, curcumin caused significant inhibition of
EGFR promoter activity, whereas FOLFOX failed to do so (Fig.
6), suggesting 2 different mechanisms for synergistic downregula-
tion of EGFR with curcumin and FOLFOX.
The last set of experiments was carried out to determine the
potential mechanism(s) by which curcumin and/or 5-FU or FOL-
FOX inhibit IGF-1R activation. We hypothesized that this may be
due to enhanced expression of IGFBP-3, a protein that sequesters
IGFs, rendering IGFs unavailable for binding to and activation of
 CURCUMIN AND CHEMOTHERAPEUTIC AGENTS IN COLON CANCER
FIGURE 7 – Changes in (a) IGFBP-3 expression and (b) sequestra-
tion of IGF-1 by IGFBP-3 in HCT-116 and HT-29 cells in response to
curcumin, 5-FU, FOLFOX, 5-FU 1 curcumin or FOLFOX 1 curcu-
min. For determination of sequestration of IGF-1, IGFBP-3 was
immunoprecipitated with anti-IGFBP-3 antibodies and the immuno-
precipitates were subjected to Western-blot analysis with anti-IGF-1
antibodies. The numbers underneath each band represent percent of
representative control. The experiment was repeated at least 3 times.
IGF-1R. We observed that not only curcumin, but also 5-FU, cur-
cumin 1 5-FU or FOLFOX markedly increased (105–264%
increase) IGFBP3 levels in HCT-116 cells (Fig. 7a). However, the
magnitude of stimulation was greatest (452%) in response to the
combination of curcumin and FOLFOX (Fig. 7a). On the other
hand, in HT-29 cells, there was no significant increase in IGFBP3
levels in response to any of the above treatments (Fig. 7a). This
could be partly be attributed to the p53 status of HT-29 cells,
which are p53-negative. This observation is consistent with p53-
mediated IGFBP3 induction.24
To further determine if the increase in IGFBP-3 levels will
result in increased binding of IGF-1, lysates from the control and
cells treated with different agents/regimen were subjected to
immunoprecipitation with IGFBP-3 antibodies, and the immuno-
precipitates were analyzed by Western-blot using anti-IGF-1 anti-
body. As shown in Figure 7b, the amount of IGF-1 bound to
IGFBP-3 was highest in response to curcumin and FOLFOX com-
bination than that noted with curcumin, 5-FU or FOLFOX treat-
ment. This was observed only in HCT-116 but not in HT-29 cells.
These observations suggest that synergistic inhibition of IGF-1R
with curcumin and FOLFOX combination may be due to increase
in IGFBP3 levels, which in turn sequester IGF-1, thus preventing
IGF-1R activation.
Discussion
Curcumin [diferuloylmethane; 1,7-bis-(4-hydroxy-3-methoxy-
phenyl)-1,6-heptadiene-3,5-dione], the major pigment in turmeric
powder, has been shown to prevent adenoma development in the
intestinal tract of Min/1 mice and inhibit chemically induced car-
cinogenesis in the colon when administered during initiation and/
or postinitiation phases.14–17 Curcumin’s chemopreventive effec-
tiveness has also been documented during the promotion/progres-
sion phases of colon carcinogenesis.25,26 One of the important
mechanisms of prevention of carcinogenesis by curcumin is down-
regulation of several cytochrome p-450 enzymes and induction of
phase II metabolizing enzymes such as glutathione S-transferase
translating into decreased M1G DNA adduct formations.27,28 Cur-
cumin’s effect on progression of the cancer is pleotropic.13,29
271
Plethora of evidences suggests that the chemopreventive effect is
mainly due to inhibition of COX-2, which leads to inhibition of
prostaglandin E2 and subsequent colon cancer growth.13,25 COX-
2 inhibition is due to attenuation of activation of transcription fac-
tors AP-1 and NF-jB, which regulate its transcription.25,30 The
mechanism of inhibition of these transcription factors is less clear.
There is some evidence that curcumin inhibits ligand-induced acti-
vation of EGFR and decreases its transcription via inhibition of
erg-1 transcription factor,31 although its effect on other EGFRs
and IGF-1R is not well studied. Curcumin has also been shown to
be an effective inhibitor of cell growth in prostatglandin-synthesis
deficient cancer cells (HCT-15), suggesting that it may also act via
prostaglandin independent pathways.12
Our current data, for the first time, demonstrate that curcumin
acts synergistically with FOLFOX in inhibiting the growth of co-
lon cancer cells. This could be attributed to attenuation of not only
EGFRs but also IGF-1R activation and their subsequent down-
stream signaling. The latter is evidenced by the observation that
expression and activation of AKT are decreased. This is in agree-
ment with our previous observation, where we demonstrated that
curcumin in combination with EGFR-Related Protein (ERRP)
causes a greater growth inhibition of colon cancer cells through
attenuation of EGFR and IGF-1R signaling. Moreover, expression
of COX-2 was maximally inhibited by the combination of curcu-
min and FOLFOX, and the fact that all gastro-intestinal cancers
show increased expression of COX-232 further supports the con-
tention that the current therapeutic strategy targets critical intracel-
lular regulators of cancer cell growth.
Abnormal activity of EGFRs has been associated with the de-
velopment and progression of many malignancies, including that
of the colon.33,34 In particular, overexpression of EGFR and HER-
2 in colorectal cancers correlates with an extremely poor clinical
prognosis.35,36 A majority of solid tumors, including those of the
colon, overexpress one or more members of the EGFR and coex-
pression of EGFR with HER-2 or HER-3 results in the develop-
ment of enhanced drug resistance.37,38 These tumors would thus
escape treatment with drugs that target only one member of the
EGFR family. The fact that curcumin together with FOLFOX tar-
gets not only EGFR but also HER-2 and HER-3 underscores the
superior value of this therapeutic regimen. The fact that curcumin
but not FOLFOX decreased activation of EGFR promoter suggests
that synergistic downregulation of EGFR in response to curcumin
and FOLFOX could be the result of 2 different mechanisms: tran-
scriptional and nontranscriptional.
Another surface growth factor receptor that is gaining impor-
tance in colon cancer is IGF-1R, which is a transmembrane hetero-
dimer consisting of two-a and two-b subunits linked by disulfide
bridges. The receptor has two major ligands, IGF-1 and IGF-2.
The biologic activities of these ligands are negatively controlled
by a family of high affinity IGF-binding proteins (IGFBPs), of
which IGFBP-3 is the most important, since it can sequester IGFs
in an inactive form in the serum.39 Epidemiologic studies have
demonstrated that an increase in circulating IGF-1 and decreased
IGFBP-3 levels are frequently associated with the development of
several types of epithelial cancers, including colorectal cancer.40
Furthermore, it has been demonstrated that blockade of the IGF/
IGF-1R axis by a soluble inhibitor of IGF-1R attenuates IGF-1-
induced Akt activation and inhibits growth of human colon cancer
xenografts in mice.11 Therefore, targeting the IGF-1/IGF-1R sig-
naling pathway may be another effective strategy for the preven-
tion and treatment of colorectal cancer. Our present study shows
for the first time that curcumin in combination with FOLFOX is
very effective in inhibiting IGF-1R activation. This appears to be
due to stimulation of expression of IGFBP3 resulting in increased
sequestration of IGFs. The increased IGFBP3 expression appears
to be restricted to p53-positive HCT-116 cells suggesting a role
for this tumor suppressor gene in regulating IGFBP3. Consistent
with this observation, we also noted relatively less apoptosis in
p53-negative HT-29 cells in response to chemotherapeutic agents/
regimen, alone or in combination with curcurmin.
272 PATEL ET AL.
Colorectal cancers are believed to develop through 2 distinct
genetic pathways namely microsatellite instable (MSI) geno-
type and microsatellite stable genotype (MSS).41,42 These 2
pathways diverge prior to development of SMAD4 mutations
and chromosomal instabilities.43 MSI is a result of defective
DNA mismatch repair pathway and is responsible for 15–20%
of the sporadic and about 85–95% of hereditary nonpolyposis
colorectal cancers. MSI is associated with a high incidence of
second primary colon cancer.5 Two of the colon cancer cells
studied here are different with regard to the microsatellite sta-
ble genotype. HCT-116 is MSI, whereas HT-29 is MSS.38 Our
results clearly show that the effect of curcumin either alone
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