Immunomodulatory effects of curcumin: In-vivo

Abstract

Curcumin specifically exhibits cytostatic and cytotoxic effects against tumors of multiple origin. Previously we have demonstrated apoptotic activity of curcumin against tumor cells with no effect on normal cells in-vitro. Many anti-cancer drugs exhibit deleterious effects on immune cells, which restrict their wide use in-vivo. In the present study, we have evaluated the effect of curcumin on the major functions of T cells, natural killer cells, macrophages and on total splenocytes in-vivo, which insight the role of curcumin on their broad effector functions. This study demonstrates that prolonged curcumin-injections (i.p.) do not impair the cytotoxic function of natural killer cells, the generation of reactive oxygen species and nitric oxide from macrophages and the levels of Th1 regulatory cytokines remained unaltered. Interestingly, curcumin-injections enhanced the mitogen and antigen induced proliferation potential of T cells. We have also evaluated immunomodulatory effects of curcumin in ascites-bearing animals. This study strengthens our belief that curcumin is a safe and useful immunomodulator for the immune system.

Full text

Immunomodulatory effects of curcumin: In-vivo
Ch. Varalakshmi, A. Mubarak Ali, B.V.V. Pardhasaradhi,
Raghvendra M. Srivastava, Sarvjeet Singh, Ashok Khar ⁎
Centre for Cellular and Molecular Biology, Uppal Road, Hyderabad 500 007, India
Received 21 September 2007; received in revised form 11 January 2008; accepted 11 January 2008
Abstract
Curcumin specifically exhibits cytostatic and cytotoxic effects against tumors of multiple origin.
Previously we have demonstrated apoptotic activity of curcumin against tumor cells with no
effect on normal cells in-vitro. Many anti-cancer drugs exhibit deleterious effects on immune
cells, which restrict their wide use in-vivo. In the present study, we have evaluated the effect of
curcumin on the major functions of T cells, natural killer cells, macrophages and on total
splenocytes in-vivo, which insight the role of curcumin on their broad effector functions. This
study demonstrates that prolonged curcumin-injections (i.p.) do not impair the cytotoxic
function of natural killer cells, the generation of reactive oxygen species and nitric oxide from
macrophages and the levels of Th1 regulatory cytokines remained unaltered. Interestingly,
curcumin-injections enhanced the mitogen and antigen induced proliferation potential of T cells.
We have also evaluated immunomodulatory effects of curcumin in ascites-bearing animals. This
study strengthens our belief that curcumin is a safe and useful immunomodulator for the immune
system.
© 2008 Elsevier B.V. All rights reserved.
KEYWORDS
Curcumin;
NO;
Lymphoproliferation;
ROS;
Macrophages;
NK cells
1. Introduction
Cancer chemotherapy is often associated with the side-
effects on immune cells. Thus, the prerequisites for anti-
cancer drugs are to ensure no damaging effects on the
immune cells, failing which the drug may completely
terminate the subsided immune response in tumor-bearing
host.
Curcumin is an illustrious dietary ingredient of the Indian
subcontinent [1]. In solution, curcumin [1,7-bis (4-hydroxy-
3-methoxyphenyl)-1,6-heptadiene-3, 5-dione] exists as a
keto-enol tautomer, which is extracted from the rhizome of
Curcuma longa [2]. Curcumin is a yellow, hydrophobic
fluorescent molecule, which rapidly impregnates in cellular
membranes [3,4].
Overwhelming evidences have confirmed that curcu-
min is an anticancerous compound and executes its
function by modulating multiple targets in tumor cells
[5,6]. Inhibition of NF-κB activation before IκBαphos-
phorylation [7], ROS (Reactive Oxygen Species) genera-
tion in tumors is the major target of curcumin, which
induce tumor cell apoptosis [8].Ontheotherhand,
carcinogen mediated tumor initiation is prevented by
Abbreviations: Ag, Antigen; CsA, CyclosporinA; ConA, Concana-
valinA; PHA, Phytohaemagglutinin; ROS, Reactive oxygen species;
NO, Nitric oxide; Mϕ, Macrophages.
⁎Corresponding author. Fax: +91 40 27160591, 27160311.
E-mail address: khar@ccmb.res.in (A. Khar).
1567-5769/$ - see front matter © 2008 Elsevier B.V. All rights reserved.
doi:10.1016/j.intimp.2008.01.008
www.elsevier.com/locate/intimp
International Immunopharmacology (2008) 8, 688–700

curcumin, involving ROS, reactive nitrogen species; NF-κB,
(NF-E2)-related factor 2, heme oxygenase-1, glutathione S-
transferase and glutathione reductase [9].In-vivo curcu-
min inhibited the generation of intestinal tumors by mod-
ulating intestinal immune cell profile [10].
In-vitro the apoptotic activity of curcumin on tumor
cells differs with dose. We have reported that induction
of stress response rendered tumor cell lines resistant to
curcumin-mediated apoptosis, which was dependent on
ROS intermediates [8]. Curcumin dose directly deter-
mines ROS generation capacity, intracellular ATP levels,
apoptosis or necrosis in osteoblast [11].Curcuminhas
also been shown to modulate the reversal of multi-drug
resistance [12].
We have previously reported the involvement of ROS in
curcumin-mediated tumor cell apoptosis leading to the
regression of ascitic tumors [13]. Curcumin also accelerated
the spontaneous regression of solid tumors involving mod-
ulation of innate immune response [14].Inaddition,
depletion of endogenous glutathione enhanced the sensitiv-
ity of tumor cells to curcumin and curcumin had no effect on
normal hepatocytes [15].
The direct role for curcumin has also been demonstrated
in the cure of various autoimmune disorders, curcumin
inhibited IL-12 mediated Th1 dependent neuronal demyeli-
nation in murine model of multiple sclerosis by targeting
Janus kinase 2, tyrosine kinase 2, STAT3 and STAT4 [16].
Curcumin also enhanced the clearance of amyloid-β(pla-
ques) in the brain by Mϕs (macrophages) in Alzheimer's
patients [17]. Under severe conditions of infection, curcumin
attenuated LPS-mediated endotoxemia [18]. Curcumin tar-
gets TLR-adapter-MD-2 and inhibits homodimerisation of
TLR4 to exhibit anti-inflammatory response [19,20]. Curcu-
min has been shown to attenuate the expression of IL-1β,
IL-6, and cyclin E in TNF-αtreated human keratinocytes
[21]. Curcumin also prevented rheumatoid arthritis, by
inducing apoptosis and inhibiting prostaglandin E2 produc-
tion in synovial fibroblasts of rheumatoid arthritis patients
[22,23]. Curcumin controls allergic responses by attenuat-
ing Th2 inflammatory responses [24].
In plasma 2.25 µg/ml of curcumin concentration can be
achieved within 15 min, by injecting 0.1 g/kg of curcumin
(i.p.) in mice. Similarly 26.06 µg/g of curcumin was found
in spleen after 1 h of injections [25]. In circulation the
major curcumin biotransformants are curcumin-glucuro-
nide, sulfate, hexahydrocurcumin, hexahydrocurcuminol,
and hexahydrocurcumin glucuronide. Synthetic curcumin
derivatives differ in their apoptotic and redox regulatory
activities [26].
It is plausible that customary consumption of curcumin
does not exert health disorders in humans; moreover it
exhibits a promising role in various pathological conditions
ranging from cancer to autoimmune disorders and inflamma-
tion. Owing to multiple mechanisms and distinct responses
on various cell types, it becomes imperative to assess its
effect on immune cells in-vivo. This study was carried out to
investigate the role of curcumin on the major functions of
the immune cells in-vivo. We have studied the effect of
curcumin on T cell proliferation, NK cell mediated cytotoxi-
city, production of cytokines, generation of NO and ROS by
Mϕs, which are the major effectors of anti-tumor immune
response in-vivo.
2. Materials and methods
2.1. Animals and cell lines
4–6 week old inbred strains of Wistar rats (females; 100–120 gm)
were used for this study, and all animal experiments were done
following the animal ethics committee and institutional guidelines.
YAC-1 (Murine lymphoma), CHO (Chinese hamster ovary), F111 (rat
fibroblast cell line), NIH3T3 (Swiss mouse embryo cells), HCE (human
corneal epithelial), RSF (rat skin fibroblast), primary cultures of
lymphocytes, hepatocytes and human tumor cell lines HL-60,
OVCAR-8, MDAMB, HepG2 were maintained in RPMI-1640 medi-
um (Gibco-BRL, Life Technologies Ltd), with 10% fetal calf serum,
100 U/ml penicillin and 50 μg/ml streptomycin (Sigma Chemical Co.,
St. Louis, MO).
2.2. Curcumin and Cyclosporin A administration
Wistar rats (8 animals/group) were given curcumin-injections
(40 mg/kg/day, i.p.) for 30 days at the intervals of 24 h or injected
with PBS, which was used as curcumin vehicle. In another 4 distinct
groups of experimental rats; Curcumin, Curcumin + Cyclosporin A,
Cyclosporin A and vehicle were injected (i.p.). In these rats cur-
cumin (40 mg/kg/rat/24 h for 30 days) was injected; Cyclosprin A
(10 mg/kg, i.p.) was injected 48 h prior to sacrificing the animal.
2.3. Collection of serum
Retro-orbital bleeding was performed to obtain blood from experi-
mental rats, which was subjected to coagulation and separation of
the sera. The sera were stored at −20 °C for various experiments.
2.4. Antibodies and reagents
Hybridomas mouse anti-rat OX-62 mAb (αE integrin), anti-IL-12-p7O
(clone C17.5) and anti-IFN-γantibody (clone XMG 1.2) were kindly
provided by Dr M.J. Puklavec, Dr G. Trincheri and Dr R.L. Coffman
respectively. Anti-OX-41 mAb was from Serotec, mAb 3.2.3 (specific
for rat NKRP-1 receptor, Endogen Inc., USA), anti-CD3 (clone 145-
2C11) and anti-IL-2 (clone HB-8794) are hybridoma clones. Anti-
CD45R antibody was from BD Pharmingen. Curcumin and CsA
(Cyclosporin A) were purchased from Sigma. Anti-mouse Ig Alexa-
488 was purchased from Molecular Probes. Anti-rat HRPO was pro-
cured from Amersham International UK. Con-A (Concanavalin-A)
and PHA (Phytohaemagglutinin) were purchased from Pharmacia
Biotech.
2.5. Preparation of splenocytes and their fractionation
Spleens from experimental rats were teased in cold PBS and total
splenocytes were obtained by Ficoll-Hypaque density gradient
centrifugation. RBCs were removed by hypotonic shock (0.84%
ammonium chloride) and total splenocytes were used for various
experiments. Firmly attached splenic Mϕs (OX-41
+
) were obtained by
their plastic adherence (37 °C, 45 min) properties.
2.6. Tumor cell apoptosis assay and flow cytometry
Induction of apoptosis in tumor cells and normal cells was assessed
by flow cytometry after propidium iodide staining of permeabilized
fixed cells. Briefly, the cells were fixed in 80% methanol for 20 min
and washed with PBS. Afterward, the cells were stained with
propidium iodide (0.05 mg/ml, propidium iodide in 0.1% sodium
citrate, 0.3% NP-40, 0.02 mg/ml RNAse for 30 min). Stained cells
were analyzed for their cell cycle stage by FACS and data were
analyzed using cell quest software.
689Effect of curcumin on immune cells

Splenocytes were fixed in 1% paraformaldehyde (15 min) and
washed with PBS. Cells were incubated with pre-standardized con-
centrations of specific mAb for 90 min, washed thrice and incubated
with appropriate secondary antibodies for 45 min (Alexa-488, Alexa-
568 or Alexa-594) in blocking solutions (1% BSA in PBS). Stained
cells were analyzed on FACS (Becton Dickinson) and the data were
analyzed using cell quest software.
2.7. Lymphoproliferation assay
Lymphocytes were prepared from the spleens of control, curcumin +
CsA and curcumin or CsA alone injected rats by Ficoll-Hypaque
gradient. 2 × 10
5
cells/well were incubated with either ConA or PHA
(0.5 to 2.5 μg/ml) for 48 h followed by the addition of [
3
H] thymidine
(1 μCi/well) and incubated further for 24 h. The cells were harvested
and the incorporated radioactivity was measured. Normal cell lines
were plated with different concentrations of curcumin (2 and 5 μM)
for 72 h, in which [
3
H] thymidine was added 12 h before harvesting.
The cells were harvested and the incorporated radioactivity was
measured in a Packard liquid scintillation counter.
As a source of tumor Ags, fixed AK-5 tumor cells (5× 10
6
cells.)
were injected s.c. in the animals (3 rats) that were given curcumin
(i.p.) injections for 30 days. Spleens were removed aseptically from
experimental animals and teased in cold PBS. Total splenocytes were
obtained by Ficoll-Hypaque density gradient, washed and plated for
30 min for macrophage (attached cells) elimination and the non-
adherent cells were collected and splenic DCs were isolated using
Dynal magnetic beads coated with anti-CD103
+
mAb (OX-62 mAb)
as per manufacturer's description (Dynal, chantilly, VA, USA). The
isolated cells were plated in IMDM-FCS overnight at 37 °C in a
CO
2
incubator. Using the above-mentioned protocol, we obtained
~95% pure DCs as assessed by morphology and phenotypic markers
(OX-62
+
, MHC II
+
, CD86
+
). No differences were observed for MHCII
expression on DCs obtained from the control and curcumin-injected
animals.
Similarly, CD3
+
T cells (anti-CD3 mAb) were isolated from total
splenocytes of experimental animals and respective controls by
Dynal magnetic bead coated with specific mAbs. The purity of T cells
was N95% as assessed by FACS using anti-CD3 mAb. DCs and T cells
were incubated at (5:1 ratio) for 3 days and [
3
H]-Thymidine (1 μCi/
well) was added 12 h before harvesting. Incorporated radioactivity
was measured in a Packard liquid scintillation counter after cell
harvesting.
2.8. Cytotoxicity assay
Splenocytes from normal rats were used for NK cell isolation using
magnetic beads coated with 3.2.3 mAb. NK cells were isolated using
Dynal magnetic beads coated with 3.2.3 mAb as per manufacturer's
description. Briefly, splenocytes were incubated with 3.2.3 mAb
coated Dynal-magnetic beads for 45 min and other cell types were
washed away. Detachment of NK cells was performed by Detach-a-
bead cocktail for 90 min. (Dynal, Chantilly, VA, USA). Cytotoxicity
assay was performed by a 4 h
51
Cr-release assay. The target cells
were incubated with 250 µCi of Na
2
Cr
51
O
4
at 37 °C for 45 min with
regular shaking. The cells were washed thrice with PBS to remove
the free radioactive label. NK cells were incubated with
51
Cr-labeled
YAC-1 cells (E:T = 100:1) for 4 h.
51
Cr released into the medium
was counted in a Packard Gamma counter and the percentage
cytotoxicity was calculated, as (% Cytotoxicity = 100 ×Experimental
cpm−Spontaneous cpm / Total cpm−Spontaneous cpm).
2.9. Enzyme linked immunofiltration assay
Cytokines in the sera of “Control; Curcumin + CsA; Curcumin; or CsA”
injected animals were estimated with specific mAbs using an en-
zyme-linked immunofiltration assay (ELIFA; Pierce Chemical Co,
Rockford, IL USA). Briefly, nitrocellulose membrane (0.22 μ) was
placed in the ELIFA unit and the Ag solutions (culture supernatants or
diluted serum samples) were filtered through nitrocellulose mem-
brane, which traps the protein specifically, followed by blocking
with BSA (3%), and the membrane was treated with primary Abs. The
membrane was washed and incubated with HRPO-tagged secondary
Ab. Afterward the signal was developed using chromogenic sub-
strate, OPD (2 mg/ml in PBS; 100 μl and 6 μlofH
2
O
2
). The reaction
was stopped by adding 50 μl of 2N sulphuric acid. The signal was read
at 490 nm along with positive control. Cytokine concentrations
were extrapolated from a standard curve generated using reference
standards for each cytokine.
2.10. NO estimation
Mϕs were cultured for 16 h, 100 μl of the cell free culture super-
natants were aspirated and the NO content was measured with
Griess reagent. The absorbance at 540 nm was measured using ELISA
reader (Molecular devices, Spectra Max 190). Nitrite content was
quantified from a standard curve generated using sodium nitrite.
2.11. Estimation of extracellular ROS
Mϕs(2×10
6
cells/well) were plated in 150 μl of phenol-red free
IMDM and the release of superoxide anions were estimated in the
presence of 80 μM cytochrome cwith/without SOD (300 U/ml) [27].
Briefly, superoxide-induced reduction of ferricytochrome cwas
monitored spectrophotometrically at 550 nm. Mϕs from control
and curcumin-injected animals were plated in complete phenol red-
free IMDM medium in 96-well plates. The time-dependent super-
oxide anion release was estimated in the presence of 80-µmol/l
cytochrome cwith and without the addition of superoxide dismutase
(300 units/ml). The cellular proteins were estimated with Bradford
reagent (Bio-Rad, Hercules, CA) using bovine serum albumin as the
standard.
2.12. Estimation of intracellular ROS
Intracellular superoxide was estimated by flow cytometry, using the
oxidation sensitive fluorescent probe 2',7'-dichlorofluorescein dia-
cetate (DCF; Molecular Probes). The cells were washed with PBS and
incubated with DCF (100 μM) for 60 min at 37 °C. At different time
intervals, cells were collected and analyzed by flow cytometry.
2.13. Statistical analysis
Student's t-test was performed to analyze the differences between
control and experimental groups. Differences were assumed to be
significant at Pb0.01.
3. Results
3.1. Specificity of apoptotic induction in tumor cells by
curcumin
We first evaluated the induction of apoptosis in CHO, rat skin
fibroblasts, human corneal epithelial cells, rat lymphocytes and
hepatocytes upon curcumin treatment and curcumin failed to
induce apoptosis (Fig. 1A), whereas curcumin induced apoptosis
in a few transformed cell lines such as MDAMB (breast car-
cinoma), OVCAR-8 (ovarian carcinoma), HepG2 (hepatocellular
carcinoma) and HL-60 (leukemia cell line) (Fig. 1B). Curcumin
induced apoptosis in all these tumor cell lines while it had no
effects on primary cultures or non-transformed cells under
similar conditions. These observations suggested that curcumin
690 Ch. Varalakshmi et al.

induces apoptosis specifically in tumor cells but not in non-
transformed cells.
3.2. Curcumin enhances the proliferative capacity of T
cells
To evaluate the effect of curcumin on cellular proliferation,
primary cultures and non-transformed cell lines of distinct or-
igin were incubated with curcumin (2 μM and 5 μM) and [
3
H]
thymidine for 72 h and the incorporated radioactivity was mea-
sured (Fig. 2A). No significant differences in [
3
H]-Thymidine
incorporation between curcumin treated and controls were
observed in-vitro.
In order to check the in-vivo effect of curcumin on the
proliferation capacity of T cells, curcumin was injected in an-
imals (i.p.) for 30 days and the splenocytes from the control and
curcumin injected animals were harvested. Mitogens like PHA
and ConA are known to specifically induce the proliferation of T
cells. Hence lymphocytes from control and curcumin-injected
rats were harvested on day 30, and treated with different
concentrations of PHA (0, 1, 2.0 μg/ml) in-vitro. An enhanced
lymphoproliferative capacity of T cells was observed in cur-
cumin-injected rats (Fig. 2B). To further confirm the effect of
curcumin on T cell proliferation in-vivo, we stimulated the total
splenocytes with another mitogen ConA (0 and 2.0 μg/ml) from
the animals that had received curcumin up to days 20 and 30. As
observed with PHA, an enhanced lymphoproliferative effect of
curcumin was observed with Con-A; moreover the lymphopro-
liferative effect was more enhanced with ConA (Fig. 2C). We
have also confirmed the specific lymphoproliferative effect of
curcumin in-vivo, by using a potent immunosuppressant Cyclos-
porine A (CsA). CsA injection resulted in a marked reduction on
ConA induced proliferation of T cells harvested from curcumin-
injected animals, however, CsA also had negligible effect on
ConA induced T cell proliferation in control animals (Fig. 2D)
suggesting the specificity of curcumin induced T cell prolifera-
tion in-vivo. The enhanced Ag-specific T-cell proliferation was
also observed in curcumin-injected rats that were injected with
fixed AK-5 tumor cells as a source of tumor Ag (Fig. 2E).
3.3. Curcumin has no effect on ROS generation
by macrophages
Mϕs function locally in various tissues and tumor-associated Mϕs
are reported to participate in anti-tumor activity. Moreover Mϕs
are the major cell type that produce ROS and NO with various
Figure 1 Specificity of induction of apoptosis in tumor cells by curcumin: (A) 1 ×10
6
CHO, RSF, HCE, lymphocytes, hepatocytes were
incubated with 25 μM curcumin for 24 h. (B) 1 × 10
6
HL-60, OVCAR-8, MDAMB and HepG2 cells were incubated with 25 μM curcumin for
24 h. Cells were washed, fixed in 70% methanol and stained with propidium iodide. Percentage apoptotic cells were evaluated by flow
cytometry.
691Effect of curcumin on immune cells

stimuli. To test whether curcumin-injection in animals affects
the free radical generation by Mϕs, we measured the extra-
cellular and the intracellular ROS levels in the Mϕs from control
and curcumin-injected animals. Interestingly no effect in the
extracellular ROS levels was observed in curcumin-injected
animals in comparison to the control; this ‘null effect’of cur-
cumin was confirmed in Mϕs isolated from two distinct anat-
omical locations viz-peritoneal cavity and spleen (Fig. 3A, B). On
days 10 and 20 of curcumin-injection, increased intracellular
ROS levels were observed in the peritoneal Mϕs, whereas on day
30 the level was similar to the controls (Fig. 3C). In contrast to
high ROS levels in peritoneal Mϕs, splenic Mϕs from normal and
curcumin-injected animals did not show any significant differ-
ences in the intracellular ROS levels (Fig. 3D). High intracellular
Figure 2 Effect of curcumin on the proliferation of non-transformed cell lines in-vitro. (A) Incorporation of [
3
H] thymidine by non-
transformed cell lines upon treatment with 2 μM and 5 μM curcumin in comparison with untreated controls.(B) Incorporation of [
3
H]
thymidine in control and curcumin-injected rat splenocytes on day 30, upon stimulation with different concentrations of PHA.
(C) Incorporation of [
3
H] thymidine in control and curcumin-injected rat splenocytes on days 20 and 30, upon stimulation with Con-A.
(D) Incorporation of [
3
H] thymidine in control, curcumin, curcumin + CsA and CsA-injected rat splenocytes on days 20 and 30, upon
stimulation with 2 μg/ml of Con-A. CsA treatment results in the inhibition of curcumin-mediated enhanced lymphoproliferative
capacity of splenocytes; also curcumin-injections enhanced proliferation of rat lymphocytes. (E) Incorporation of [3H] thymidine in T
cells harvested from control (fixed AK-5 tumor cell injected) and curcumin (fixed AK-5 tumor cell) injected animals was measured.
Data shown are mean ± SD and are representative of 3 similar experiments.
692 Ch. Varalakshmi et al.

ROS in the peritoneal Mϕs on days 20 and 30, seems to reflect the
local effects of curcumin in the peritoneal cavity since similar
effect was not observed in the splenic Mϕs.
We have also assessed the intracellular ROS levels in the
peritoneal and splenic Mϕs from rats that had received the
injections of Curcumin, Curcumin + CsA or CsA alone. In the
presence of CsA, increased ROS levels were found in the peri-
toneal and splenic Mϕs on day 20 but not on day 30, which
further reflected short-term effect of curcumin (Fig. 3E, F).
However an enhanced oxidative load was also observed with CsA
alone on day 20; hence these data reflect the synergistic effect
of curcumin in conjunction with CsA on day 20.
To evaluate the modulation of ROS generation in Mϕsby
curcumin and tumor, we transplanted AK-5 tumor cells (i.p.) in
control and curcumin-injected rats (30 days curcumin treat-
ment). On day 5 after tumor transplantation, no effect of AK-5
tumor was observed on ROS levels in splenic Mϕs in comparison
to the control. Thus ROS levels upon AK-5 transplantation in
curcumin-injected rats, do not reflect any changes (Fig. 3G).
3.4. Curcumin has no effect on the NO generation
by macrophages
NO accumulation in culture supernatants was estimated after
24 h culture of Mϕs, from controls and curcumin-injected rats.
No significant differences were seen between the NO secreting
capacity of control and curcumin-injected rats for both peri-
toneal and splenic Mϕs(Fig. 4A,B). In-vitro activation of Mϕs
from both control and curcumin-injected rats showed no
differences in LPS-mediated NO production. It has been shown
in several studies that LPS-mediated NO production can be
blocked completely by curcumin in-vitro [28]. However our data
suggest that in-vivo priming of Mϕs by curcumin does not abort
LPS responsiveness. On the other hand, CsA inhibited the effect
Figure 3 Effect of curcumin-injection on ROS generation by macrophages and its modulation by AK-5 ascitic tumor. Extracellular
ROS production by control and curcumin injected rat peritoneal (A), splenic (B) macrophages on days 10, 20 and 30. Similarly
intracellular ROS levels were monitored in normal and curcumin treated rat peritoneal (C) and splenic (D) macrophages on days 10, 20
and 30. (E) Intracellular ROS generation by control, curcumin, curcumin + CsA and CsA-injected rat peritoneal macrophages. CsA
injection increased the oxidative load and hence curcumin and CsA exert a synergistic effect on intracellular ROS generation. (F)
Intracellular ROS generation by control, curcumin, curcumin + CsA and CsA-injected rat splenic macrophages. (G) Intracellular ROS
levels in control and curcumin-injected rat (30 days) splenic macrophages after transplantation of AK-5 tumor (i.p.) on day 5. Results
shown are indicative of two independent experiments under similar conditions.
693Effect of curcumin on immune cells

of LPS on NO production, which was found to be independent of
curcumin (Fig. 4C, D). AK-5 is a highly virulent ascitic tumor and
suppresses NO generation capacity of Mϕs through secretary
TGF-β1. We observed that AK-5 tumor suppresses NO generation
of Mϕs and curcumin-primed peritoneal and splenic Mϕs (30 days
curcumin treated) failed to reverse the inhibitory effect of AK-5
on Mϕs(Fig. 4E and F). We have previously shown that curcumin-
injection regressed AK-5 ascites [13]. Hence these studies
conclude that curcumin-injections in ascites-bearing animals
exert direct effect on tumor cells. These results suggest that
under in-vivo prolonged treatment conditions curcumin has no
inhibitory function on NO production by Mϕs.
3.5. Curcumin has no effect on NK cell cytotoxic potential
In order to investigate if curcumin affected the cytotoxic
capability of NK cells (Natural killer cells), we isolated NK cells
from control and curcumin-injected animals on different days
(10, 20 and 30) and assessed their cytotoxic ability against YAC-1
tumor cells in a 4 h
51
Cr release assay. Splenic NK cells from
control and curcumin-injected rats exhibited similar levels of
cytotoxicity against YAC-1 targets at 100:1 ratio (Fig. 5A).
Similar effect was observed at 50:1 and 20:1 E:T ratio (data
not shown). CsA injections abolished the cytotoxic function of
NK cells isolated from control or curcumin-injected animals
(Fig. 5B). We have previously demonstrated that curcumin
administration regressed AK-5 ascites and also accelerated the
regression of AK-5 solid tumors [14]. We further evaluated the
role of curcumin on NK cell function during the progression of
ascites tumor. A regular dose of curcumin was given to animals
for 30 days (i.p.) and then low dose (1 × 10
6
cells) of AK-5 tumor
cells were transplanted i.p. Similar to our previous observations
[13], regression of the ascitic tumors was observed (data not
shown), whereas, there was no enhancement of splenic NK cell
cytotoxic function in curcumin-injected rats on day 5, thereby
suggesting a direct role of curcumin in causing tumor eradication
(Fig. 5C).
3.6. Cytokine profile in curcumin administrated animals
The immunomodulatory effect of curcumin was also estimated in
terms of cytokine levels in the serum samples of control and
Figure 3 (continued).
694 Ch. Varalakshmi et al.

curcumin-injected animals on different days. We have quanti-
fied the levels of IL-2, IL-12 and IFN-γin the serum samples.
Overall, there was no considerable variation in IL-2 and IFNγ
levels between the control and the curcumin-injected animals
(Fig. 6A, B, C). However, IL-12 levels were slightly elevated in
curcumin-injected animals on days 10 and 20, which were
comparable to the controls on day 30 (Fig. 6A). IL-12 and IFN-γ
levels in CsA treated, control and curcumin treated animals did
Figure 4 Effect of curcumin administration on the production of NO by macrophages and its modulation by ascitic tumor. (A) NO
production by control and curcumin-injected rat peritoneal macrophages on days 10, 20, 30. (B) NO production by control and
curcumin-injected rat splenic macrophages on days 10, 20, 30. (C) NO production by control, curcumin, curcumin + CsA and CsA-
injected rat peritoneal macrophages. (D) NO production by control, curcumin, curcumin + CsA and CsA injected rat splenic
macrophages. CsA inhibits LPS induced increase in NO production. (E) NO production capacity of control and curcumin-injected rat
(30 days) peritoneal macrophages, upon transplantation of AK-5 tumor (i.p.) on day 5. (F) NO production capacity of control and
curcumin-injected mouse (30 days) peritoneal macrophages, upon transplantation of Meth-A tumor (i.p.) on day 5. Values shown are
mean± SD and are representative of 3 similar experiments.
695Effect of curcumin on immune cells

not show any significant changes (Fig. 6D, F). However, CsA
injections in both control and curcumin injected animals caused
an equivalent decline in the circulating levels of IL-2 suggesting
that the dose of curcumin used does not interfere with the
normal production of IL-2 (Fig. 6E) and the decline in IL-2 levels
recorded is due to CsA alone. The concentration profiles of IL-12
and IFN-γin CsA injected control animals were similar to those
of CsA and curcumin-injected animals indicating that in-vivo
administration of curcumin had no effect on the cytokine levels.
4. Discussion
Curcumin has been shown to exert potent anticancerous
effects. The pharmacological safety of curcumin can be
envisaged by its regular consumption by humans as a food
additive. Curcumin exhibits distinct biological effects by
regulating the expression of distinct genes and proteins [1],
and its ‘multifaceted’action needs reasonable evaluation
before establishing it as a ‘drug’, specially for the immune
cells. Curcumin failed to exert any effect on GSH levels in
healthy animals in contrast to its inhibitory effects in car-
cinogenesis [29]. Although the anti-tumor activity of
curcumin is clinically accomplished, its immunomodulatory
effects are inadequately understood. Few studies have re-
ported immunomodulatory effects of curcumin in-vitro [30],
and we have analyzed the effects of curcumin on various
lymphocyte functions viz., mitogen and Ag-induced T cell
proliferation, NK cell-mediated cytotoxicity, ROS and NO
generation by Mφand production of cytokines, using rat as
model.
Our data show that treatment of various tumor cell lines
viz., HL-60 (acute promyelocytic leukemia), MDAMB (breast
carcinoma), HepG2 (hepatocellular carcinoma) with curcu-
min, induced apoptosis while it had no effects on primary
cells or nontransformed cells that indicate the pharmacolo-
gical safety of curcumin. Similar to our observations, no
affect on the viability of splenocytes was observed upon
treatment with low to high concentration of curcumin (10–
30 μmol/l) for 4 days [31].
Earlier studies have shown that curcumin aborted mito-
gen-induced T cell proliferation in-vitro [32], our data
Figure 5 Effect of curcumin administration on NK cell cytotoxicity and its modulation by ascitic tumor. (A) Splenic NK cells from
control and curcumin-injected animals did not show any significant differences in cytotoxic ability against YAC-1 tumors over a 30-day
period in-vitro. (B) Cytotoxic potential of splenic NK cells from control, curcumin, curcumin + CsA, and CsA-injected rats. Our
observations suggest that CsA induced decline in NK cell cytotoxicity is independent of curcumin-injection. (C) Cytotoxic potential of
control and curcumin-injected rat (30 days) splenic NK cells, upon transplantation of AK-5 tumor (i.p.) on day 5. Values shown are
mean± SD and are representative of 3 similar experiments.
696 Ch. Varalakshmi et al.

suggest that ‘curcumin-conditioning’of T cells endows ad-
ditional proliferation capacity in-vivo. Often tumors lead to
the depletion of T cells by various mechanisms. Our data
show that curcumin-injections enhance T cell proliferation
capacity in the long term, which may restore the number of T
cells in-vivo in tumor-bearing hosts. A recent study has
indeed demonstrated that tumor induced depletion of Tcells
can be reversed by dietary curcumin in-vivo. Also dietary
curcumin restores progenitor, effector, and circulating Tcells
in tumor-bearing hosts [33].Inin-vitro studies, it was dem-
onstrated that CsA aborts PHA and PMA mediated T cell
proliferation [34], and curcumin does not influence the
action of CsA in-vivo [35]. In this study we show that CsA,
aborts curcumin-mediated T cell proliferation capacity in-
vivo. In-vitro curcumin enhanced ConA mediated T cells
proliferation at low dose whereas a similar effect was not
seen with IL-2 or alloantigen mediated T cell prolifera-
tion [31]. In the same study an inverse effect of curcumin
Figure 6 Cytokine profile in curcumin injected animals. Levels of IL-12 (A), IL-2 (B) and IFN-γ(C) in control and curcumin injected
animals on days 10, 20 and 30. Levels of IL-12 (D), IL-2 (E) and IFN-γ(F) were also determined when CsA alone or in combination with
curcumin was administered in animals. CsA caused a decline in curcumin induced IL-12 levels. On the other hand, curcumin had no
effect on IL-2 and IFN-γlevels. (In Figure optical density 1= 100 Units =250 pg/ml of cytokines, which was compared with the standard
plot). Values shown are mean± SD and are representative of 3 similar experiments.
697Effect of curcumin on immune cells

concentration on lymphoproliferation upon ConA stimulation
was reported where a low concentration (6 μM) caused an
increase in the proliferative capacity and a higher concen-
tration (13 μM) led to an inhibitory effect [31]. Another study
has also reported that dietary turmeric Curcuma xanthor-
rhiza (sp) enhances blastogenic effect of mitogens [36].
These studies suggest a specific molecular mechanism for
curcumin-mediated T cell proliferation. Another report
showed that dietary curcumin also enhances colon epithelial
cell proliferation in-vivo, which shows similar effect of
curcumin on non-immune cells [37]. Moreover these results
provide the advantageous effects of curcumin in-vivo.
In our study lymphoproliferative effect of curcumin is
confirmed by the observation that in presence of CsA,
curcumin-injected animals showed higher lymphoprolifera-
tive ability than control animals. This clearly suggests that
curcumin antagonizes the lymphosuppressive effects of CsA
and curcumin administration indeed increases lymphoproli-
feration. In transgenic C57BL/6J-Min/+ (Min/+) mice, which
contain a germline mutation in Apc and serve as a model for
familial adenomatous polyposis exhibits a marked tendency
for spontaneous adenoma formation. Upon curcumin-injec-
tions in these animals a retarded growth of adenoma was
observed and an increase in intestinal CD4
+
Tcells was found,
which also correlates very well with the fact that curcumin-
mediated Tcell proliferation can inhibit tumor growth [10].A
low to high concentration of curcumin (5, 50, 250 μg/ml)
enhances ConA mediated Na/K
+
ATPase and Ca
++
ATPase
activity upon stimulation for 7 days, but not on days 3 and 5.
This finding may imply the reshuffling of receptors asso-
ciated with these transporters [38]. Curcumin also modulates
surface p-glycoprotein differentially [39,40], which is also a
target to break multiple drug resistance by curcumin [41].
Hence modulation of glycoproteins may be responsible for
the high proliferation capacity of T cells by curcumin. It is
modestly conceivable because curcumin-mediated enhanced
Tcell proliferation was not found with IL-12 or Ags in-vitro by
others [31]. In contrast, our result shows that curcumin
conditioning of Tcells can also enhance the Ag-specific T cell
proliferation. At present it is difficult to explain this action of
curcumin unambiguously, since in-vivo being a complex
system and curcumin acts on various targets. Hence the
mechanism behind the enhanced proliferation of T cells by
curcumin needs to be investigated, which can be targeted to
explore curcumin based or combinatorial therapy.
Mϕs are phagocytic cells that release ROS in response to
various stimuli. The enzyme responsible for the production
of superoxide and hydrogen peroxide is a multi-component
NADPH oxidase that requires assembly at the plasma mem-
brane to function as an oxidase [42]. Curcumin is an an-
tioxidant and exerts pro-oxidant properties under certain
conditions to induce apoptosis in tumor cells. High concen-
trations of Curcumin (~50 μM) promote ROS generation [13],
while low curcumin (~10 μM) aborts ROS generation [43].
Both antioxidant and prooxidant activities are believed to be
involved in the anticancer activity of Curcumin [44]. Kang et
al. have shown that histone acetyltransferase is one of the
in-vivo targets of curcumin. Curcumin may induce histone
hypoacetylation in-vivo, where the ROS generation may
play an important role. However only high concentrations of
curcumin cause ROS generation and inhibition of histone
acetyltransferase (HAT) activity (IC
50
for HAT activity be-
tween 25 and 50 μM), hence further studies related to the
modulation of histone acetyltransferase may be relevant to
explore the mechanisms of curcumin underlying its antic-
ancer properties in-vivo [45]. The increase in the intracel-
lular ROS levels in peritoneal Mϕs on days 10, 20 but not
on day 30 seems to be a sustained local effect of curcumin.
CsA is known to increase the oxidative stress and cause a
decrease in the reduced glutathione content and also reduce
the catalase and superoxide dismutase [46,47] activity in
the cells. CsA treatment induced an additive effect on ROS
generated intracellularly in Mϕs from curcumin-injected
animals. The synergistic effect of CsA and curcumin was
evident in both splenic and peritoneal Mϕs. The role of ROS
levels in the generation of cytokines such as IL-12 by Mϕsis
clear since intracellular ROS is known to regulate IL-12
production [48]. Curcumin also retained the property to
induce ROS generation in splenic Mϕs as observed in our
experiments.
NO is a tumoricidal molecule that induces tumor cell
killing by down regulating cyclin D1, inhibition of vital en-
zymes essential for tumor cell growth and through the
activation of caspases [49–50]. In-vivo Mϕs serve as NO
generators, moreover tumor associated Mϕs directly induce
tumor cell apoptosis through NO. Hence in-vivo effect of
curcumin on NO generation capacity of Mϕs was evaluated.
Curcumin aborts LPS-mediated NO generation in activated
Mϕsin-vitro [28], in continuous conjunction. However cur-
cumin only acts as a reversible inhibitor of iNOS (unpublished
observation). The direct target of curcumin, TLR-MD-2 has
been reported on Mϕs that participates in the inhibitory
action of curcumin in-vitro [19,20]. Our data show that in-
vivo priming of Mϕs retains the LPS responsiveness since LPS
could activate the macrophages to secrete NO. However
tumor suppressive effect on NO generation by splenic Mϕs
could not be reversed by curcumin. Our earlier data on
faster regression of solid tumor showed involvement of
splenic Mϕs in curcumin-injected animals. However the
differential effect of curcumin may also be dependent on
the site of tumor or tumor aggressiveness, which is different
for s.c. and i.p. tumors, since Mϕs differentially modulate
their function depending on the site of tumor transplanta-
tion for efficient tumor regression [14,51,52]. Moreover the
status of the tumor exhibits a direct correlation with Mϕs
activation and their migration to distinct anatomical loca-
tions in the body [53]. The curcumin independent NO in-
hibitory effect of CsA treatment on Mϕs strongly suggests
that curcumin has little or no effect, either alone or in
combination with a NO modulatory compound like CsA, on
NO generation by Mϕs. CsA also modulates the NO syn-
thesizing capacity of Mϕs[54].
NK cells are the major mediators of the innate anti-tumor
immune responses and eradicate tumors by recognition of
stress inducible ligands on tumors and execute tumor cell
killing by perforin and granzyme in-vivo [55]. NK cells erad-
icate solid tumors by apoptosis [55], whereas their function is
dependent on the tumor load in the peritoneum [56].
Curcumin augments cytotoxic potential of NK cells in-
vitro, which can be further enhanced by IFNγ[32]. Our data
show that curcumin does not enhance NK cytotoxic potential
in-vivo till day 30. Curcumin reversed breast tumor medi-
ated immune suppression of NK cell cytotoxicity [57] and
curcumin-injection enhanced regression of histiocytic solid
698 Ch. Varalakshmi et al.

tumor where NK cells participated as the primary effectors
in spontaneous regression [14].
Given that curcumin exerts either null or beneficial
effects on cellular immune functions in-vivo, we also looked
at the circulating levels of cytokines in the sera of curcumin-
injected animals, viz. IL-2 and IFN-γ, there was no increase
in the serum levels of the cytokines analyzed, whereas
slightly higher IL-12 levels in curcumin injected animals
could be explained by the fact that in curcumin injected
animals a higher ROS generation could lead to a higher IL-12
generation. At the same time the potential effects of in-
creased IL-12 levels within the animal in the form of an
increased NK cell cytotoxicity or increased T cell response
were not observed. This reflects that this increase in IL-12
levels is not sufficient to exert its biological effect in-vivo.
However, some studies have reported that curcumin inhibits
IL-12 and IL-2 production by Mϕs and T lymphocytes in-vitro
[58], again in these studies the cytokine inhibitory action of
curcumin is seen at 20 μM or above while at 5 μM or lower
concentrations the inhibitory effects were not observed. This
further validates that curcumin administration in normal
animals is safe from an immunological point of view. Fur-
thermore, curcumin did not alter the distribution of
lymphocyte subsets like NK, T, B, DC and Mϕs in the spleen
(data not shown) indicating that the increase in ex vivo
proliferation is the result of functional modifications of
effector mechanisms, since no changes in the immune cell
profile were observed in the spleen.
Data presented in this study insight that curcumin could
modulate adaptive arm of the immunity by potentiating the
proliferative ability of T cells. Nevertheless curcumin also
has some effects on the innate immune population. En-
hanced proliferative capacity of T cells indicates the al-
tered threshold of T cell activation by curcumin-injections
while it has little effect with respect to Mϕs or NK cell
activation and has no effect on circulating cytokines. These
findings suggest that the in-vivo effects of curcumin on
immune cells could be acceptable thereby establishing its
‘pharmacological safety’. It equips the immune cells with a
better proliferative capacity that should result in a stronger
immune response under pathological conditions. Our stud-
ies also suggest that curcumin is a safe pharmacological
molecule for immune cells in-vivo. This study provides the
experimental evidences that curcumin has beneficial effects
on immune cells, which is highly desirable in anti-cancer
therapy.
Acknowledgements
The authors thank Ms. T. Hemalatha for secretarial help. Mr.
N. Dwarakanath helped in animal handling. RMS and SS are
supported by SRF scholarships from the CSIR.
References
[1] Aggarwal BB, Sundaram C, Malani N, Ichikawa H. Curcumin: the
Indian solid gold. Adv Exp Med Biol 2007;595:1–75.
[2] Payton F, Sandusky P, Alworth WL. NMR study of the solution
structure of curcumin. J Nat Products 2007;70:143–6.
[3] Ingolfsson HI, Ii RE, Andersen OS. Curcumin is a modulator of
bilayer material properties. Biochemistry 2007;46:10384–91.
[4] Jaruga E, Sokal A, Chrul S, Bartosz G. Apoptosis-independent
alterations in membrane dynamics induced by curcumin. Exp
Cell Res 1998;245:303–12.
[5] Aggarwal BB, Shishodia S. Molecular targets of dietary agents
for prevention and therapy of cancer. Biochem Pharmacol
2006;71:1397–13421.
[6] Lin JK. Molecular targets of curcumin. Adv Exp Med Biol
2007;595:227–43.
[7] Singh S, Aggarwal BB. Activation of transcription factor NF-
kappa B is suppressed by curcumin (diferuloylmethane). J Biol
Chem 1995;270:24995–5000.
[8] Khar A, Ali AM, Pardhasaradhi BV, Varalakshmi CH, Anjum R,
Kumari AL. Induction of stress response renders human tumor
cell lines resistant to curcumin-mediated apoptosis: role of
reactive oxygen intermediates. Cell Stress Chaperones 2001;6:
368–76.
[9] Thangapazham RL, Sharma A, Maheshwari RK. Multiple mole-
cular targets in cancer chemoprevention by curcumin. AAPS J
2006;8:E443–9.
[10] Churchill M, Chadburn A, Bilinski RT, Bertagnolli MM. Inhi-
bition of intestinal tumors by curcumin is associated with
changes in the intestinal immune cell profile. J Surg Res
2000;89:169–75.
[11] Chan WH, Wu HY, Chang WH. Dosage effects of curcumin on cell
death types in a human osteoblast cell line. Food Chem Toxicol
2006;44:1362–71.
[12] Limtrakul P. Curcumin as chemosensitizer. Adv Exp Med Biol
2007;595:269–300.
[13] Khar A, Ali AM, Pardhasaradhi BV, Begum Z, Anjum R. Antitumor
activity of curcumin is mediated through the induction of
apoptosis in AK-5 tumor cells. FEBS Lett 1999;445:165–8.
[14] Bhaumik S, Jyothi MD, Khar A. Differential modulation of nitric
oxide production by curcumin in host macrophages and NK
cells. FEBS Lett 2000;483:78–82.
[15] Syng-Ai C, Kumari AL, Khar A. Effect of curcumin on normal and
tumor cells: role of glutathione and bcl-2. Mol Cancer Ther
2004:1101–8.
[16] Natarajan C, Bright JJ. Curcumin inhibits experimental allergic
encephalomyelitis by blocking IL-12 signaling through Janus
kinase-STAT pathway in T lymphocytes. J Immunol 2002;168:
6506–13.
[17] Zhang L, Fiala M, Cashman J, Sayre J, Espinosa A, Mahanian M,
et al. Curcuminoids enhance amyloid-beta uptake by macro-
phages of Alzheimer's disease patients. J Alzheimer's Dis
2006;10:1–7.
[18] Chen HW, Kuo HT, Chai CY, Ou JL, Yang RC. Pretreatment of
curcumin attenuates coagulopathy and renal injury in LPS-
induced endotoxemia. J Endotoxin Res 2007;13:15–23.
[19] Gradisar H, Keber MM, Pristovsek P, Jerala R. MD-2 as the target
of curcumin in the inhibition of response to LPS. J Leukoc Biol
2007;82:968–74.
[20] Youn HS, Saitoh SI, Miyake K, Hwang DH. Inhibition of ho-
modimerization of Toll-like receptor 4 by curcumin. Biochem
Pharmacol 2006;72:62–9.
[21] Cho JW, Lee KS, Kim CW. Curcumin attenuates the expression of
IL-1beta, IL-6, and TNF-alpha as well as cyclin E in TNF-alpha-
treated HaCaT cells; NF-kappaB and MAPKs as potential
upstream targets. Int J Mol Med 2007;19:469–74.
[22] Funk JL, Oyarzo JN, Frye JB, Chen G, Lantz RC, Jolad SD, et al.
Turmeric extracts containing curcuminoids prevent experimen-
tal rheumatoid arthritis. J Nat Products 2006;69:351–5.
[23] Park C, Moon DO, Choi IW, Choi BT, Nam TJ, Rhu CH, et al.
Curcumin induces apoptosis and inhibits prostaglandin E2
production in synovial fibroblasts of patients with rheumatoid
arthritis. Int J Mol Med 2007;20:365–72.
[24] Kurup VP, Barrios CS, Raju R, Johnson BD, Levy MB, Fink JN.
Immune response modulation by curcumin in a latex allergy
model. Clin Mol Allergy 2007;5:1.
699Effect of curcumin on immune cells

[25] Pan MH, Huang TM, Lin JK. Biotransformation of curcumin
through reduction and glucuronidation in mice. Drug metab
dispos 1999;27:486–94.
[26] Mishra S, Kapoor N, Mubarak Ali A, Pardhasaradhi BV, Kumari
AL, Khar A, et al. Differential apoptotic and redox regulatory
activities of curcumin and its derivatives. Free Rad Biol Med
2005;38:1353–60.
[27] Pick E. Microassays for superoxide and hydrogen peroxide
production and nitroblue tetrazolium reduction using an
enzyme immunoassay microplate reader. Methods Enzymol
1986;132:407–21.
[28] Brouet I, Ohshima H. Curcumin, an anti-tumour promoter and
anti-inflammatory agent, inhibits induction of nitric oxide
synthase in activated macrophages. Biochem Biophys Res
Commun 1995;206:533–40.
[29] Okazaki Y, Iqbal M, Okada S. Suppressive effects of dietary
curcumin on the increased activity of renal ornithine decar-
boxylase in mice treated with a renal carcinogen, ferric
nitrilotriacetate. Biochim Biophys Acta 2005;1740:357–66.
[30] Jagetia GC, Aggarwal BB. “Spicing up”of the immune system by
curcumin. J Clin Immunol 2007;27:19–35.
[31] Gao X, Kuo J, Jiang H, Deeb D, Liu Y, Divine G, et al. Immu-
nomodulatory activity of curcumin: suppression of lymphocyte
proliferation, development of cell-mediated cytotoxicity, and
cytokine production in vitro. Biochem Pharmacol 2004;68:
51–61.
[32] Yadav VS, Mishra KP, Singh DP, Mehrotra S, Singh VK. Immu-
nomodulatory effects of curcumin. Immunopharmacol Immu-
notoxicol 2005;27:485–97.
[33] Bhattacharyya S, Mandal D, Saha B, Sen GS, Das T, Sa G. Cur-
cumin prevents tumor-induced Tcell apoptosis through Stat-5a-
mediated Bcl-2 induction. J Biol Chem 2007;282:15954–64.
[34] Ranjan D, Johnston TD, Wu G, Elliott L, Bondada S, Nagab-
hushan M. Curcumin blocks cyclosporine A-resistant CD28
costimulatory pathway of human T-cell proliferation. J Surg
Res 1998;7:174–8.
[35] Deters M, Klabunde T, Meyer H, Resch K, Kaever V. Effects of
curcumin on cyclosporine-induced cholestasis and hypercho-
lesterolemia and on cyclosporine metabolism in the rat. Planta
Med 2003;69:337–43.
[36] Yasni S, Yoshiie K, Oda H, Sugano M, Imaizumi K. Dietary Cur-
cuma xanthorrhiza Roxb. increases mitogenic responses of
splenic lymphocytes in rats, and alters populations of the
lymphocytes in mice. J Nutr Sci Vitaminol 1993;39:345–54.
[37] Kim SJ, Hellerstein MK. Pharmacological doses of dietary
curcumin increase colon epithelial cell proliferation in vivo in
rats. Phytother Res 2007;21:995–8.
[38] Cohly HHP, Rao MR, Kanji VK, Patlolla B, Taylor A, Wilson MT,
et al. Effect of turmeric, turmerin and curcumin on Ca
2+
,
Na/K
+
ATPases in concanavalin A-stimulated human blood
mononuclear cells. International J Mol Sci 2003;4:34–44.
[39] Zhang W, Tan TM, Lim LY. Impact of curcumin-induced changes
in P-glycoprotein and CYP3A expression on the pharmacoki-
netics of peroral celiprolol and midazolam in rats. Drug Metab
Dispo: The biological fate of chemicals 2007;35:110–5.
[40] Chearwae W, Anuchapreeda S, Nandigama K, Ambudkar SV,
Limtrakul P. Biochemical mechanism of modulation of human
P-glycoprotein (ABCB1) by curcumin I, II, and III purified from
Turmeric powder. Biochem Pharmacol 2004;68:2043–52.
[41] Anuchapreeda S, Leechanachai P, Smith MM, Ambudkar SV,
Limtrakul PN. Modulation of P-glycoprotein expression and
function by curcumin in multidrug-resistant human KB cells.
Biochem Pharmacol 2002;64:573–82.
[42] Forman HJ, Torres M. Redox signaling in macrophages. Mol
Aspects Med 2001;22:189–216.
[43] Chan WH, Wu CC, Yu JS. Curcumin inhibits UV irradiation-in-
duced oxidative stress and apoptotic biochemical changes in
human epidermoid carcinoma A431 cells. J Cell Biochem
2003;90:327–38.
[44] Bhaumik S, Anjum R, Rangaraj N, Pardhasaradhi BV, Khar A.
Curcumin mediated apoptosis in AK-5 tumor cells involves
the production of reactive oxygen intermediates. FEBS Lett
1999;456:311–4.
[45] Kang J, Chen J, Shi Y, Jia J, Zhang Y. Curcumin-induced histone
hypoacetylation: the role of reactive oxygen species. Biochem
pharmacol 2005;69:1205–13.
[46] Chander V, Chopra K. Effect of molsidomine and L-arginine in
cyclosporine nephrotoxicity: role of nitric oxide. Toxicology
2005;207:463–74.
[47] Chander V, Tirkey N, Chopra K. Resveratrol, a polyphenolic
phytoalexin protects against cyclosporine-induced nephrotoxi-
city through nitric oxide dependent mechanism. Toxicology
2005;210:55–64.
[48] Kim SH, Kim J, Sharma RP. Inhibition of p38 and ERK MAP kinases
blocks endotoxin-induced nitric oxide production and differ-
entially modulates cytokine expression. Pharmacol Res
2004;49:433–9.
[49] BogdanC. Nitric oxide and theimmune response.Nature Immunol
2001;2:907–16.
[50] Farias-Eisner R, Sherman MP, Aeberhard E, Chaudhuri G. Nitric
oxide is an important mediator for tumoricidal activity in vivo.
Proc Natl Acad Sci USA 1994;91:9407–11.
[51] Mitra R, Bhaumik S, Khar A. Differential modulation of host
macrophage function by AK-5 tumor is dependent on the site of
tumor transplantation. Immunol Lett 2002;82:183–90.
[52]BhaumikS,MitraR,VaralakshmiC,KharA.Activated
macrophages migrate to the subcutaneous tumor site via the
peritoneum: a novel route of cell trafficking. Exp Cell Res
2001;266:44–52.
[53] Mitra R, Khar A. Role of reactive nitrogen intermediates and
protein nitration during immune response against a rat histiocy-
toma. Immunol Lett 2002;84:145–51.
[54] Hortelano S, Lopez-Collazo E, Bosca L. Protective effect of
cyclosporin A and FK506 from nitric oxide-dependent apoptosis
in activated macrophages. Br J Pharmacol 1999;126:1139–46.
[55] Khar A, Pardhasaradhi BV, Varalakshmi C, Ali AM, Kumari AL.
Natural killer cell as the effector which mediates in vivo
apoptosis in AK-5 tumor cells. Cell Immunol 1997;177:86–92.
[56] Das S, Khar A. Regulation of NK cell function in vivo by the
dose of tumour transplanted in the peritoneum. Immunol Lett
2002;83:133–42.
[57] Zhang HG, Kim H, Liu C, Yu S, Wang J, Grizzle WE, et al.
Curcumin reverses breast tumor exosomes mediated immune
suppression of NK cell tumor cytotoxicity. Biochim Biophys Acta
2007;1773:1116–23.
[58] Kang BY, Song YJ, Kim KM, Choe YK, Hwang SY, Kim TS. Cur-
cumin inhibits Th1 cytokine profile in CD4+ T cells by sup-
pressing interleukin-12 production in macrophages. Br J
Pharmcol 1999;128:380–4.
700 Ch. Varalakshmi et al.