Current Cancer Drug Targets, 2005, 5, 117-129 117

Induction of Apoptosis by Curcumin and Its Implications for Cancer Therapy

D. Karunagaran*, R. Rashmi and T. R. Santhosh Kumar

Cancer Biology Laboratory, Rajiv Gandhi Centre for Biotechnology, Thiruvananthapuram, Kerala 695 014 India
Abstract: Curcumin (diferuloyl methane), the yellow pigment in turmeric (Curcuma longa), is a potent
chemopreventive agent that inhibits cancer cells proliferation by arresting them at various phases of the cell
cycle depending upon the cell type. Curcumin-induced apoptosis mainly involves the mitochondria-mediated
pathway in various cancer cells of different tissues of origin. In some cell types like thymocytes, curcumin
induces apoptosis-like changes whereas in many other normal and primary cells curcumin is either inactive or
inhibits proliferation, but does not appear to induce apoptosis. These together with reports that curcumin
protects cells against apoptosis induced by other agents, underscore the need for further understanding of the
multiple mechanisms of cell death unleashed by curcumin. Tumor cells often evade apoptosis by expressing
several antiapoptotic proteins, down-regulation and mutation of proapoptotic genes and alterations in
signaling pathways that give them survival advantage and thereby allow them to resist therapy-induced
apoptosis. Many researchers including ourselves, have demonstrated the involvement of several pro and
antiapoptotic molecules in curcumin-induced apoptosis, and ways to sensitize chemoresistant cancer cells to
curcumin treatment. This review describes the mechanisms of curcumin-induced apoptosis currently known,
and suggests several potential strategies that include down-regulation of antiapoptotic proteins by antisense
oligonucleotides, use of proapoptotic peptides and combination therapy, and other novel approaches against
chemoresistant tumors. Several factors including pharmacological safety, scope for improvement of structure
and function of curcumin and its ability to attack multiple targets are in favor of curcumin being developed as a
drug for prevention and therapy of various cancers.
Keywords: Apoptosis, curcumin, Bcl-2, hsp 70, Bcl-XL, Ku70, antisense, cancer.

INTRODUCTION carcinogenesis may also promote apoptosis, thereby

producing selective pressure to override apoptosis. Finally,
Curcumin (diferuloyl methane) is a naturally occurring
it is well documented that most cytotoxic anticancer agents
yellow pigment isolated from the rhizome of the perennial
induce apoptosis, raising the intriguing possibility that
herb Curcuma longa that has been cultivated for centuries in
defects in apoptotic programs reduce treatment sensitivity
several Asian countries (India, Nepal, China and Indonesia)
and contribute to treatment failure. Hence, it is not
[1, 2]. Turmeric, the powdered form of the rhizome, is
surprising that several laboratories consider apoptosis as the
widely used in these countries in foods (spice and coloring
major mechanism by which most chemopreventive and
agent), traditional medicines (to treat a variety of ailments),
chemotherapeutic agents act in arresting cell cycle
textile and pharmaceutical industries as well as in Hindu
progression and/or triggering programmed cell death [13-15].
religious ceremonies [3-5]. Curcumin possesses
Apoptosis is accompanied by certain well-characterized
antiinflammatory and antioxidant properties [6-8]. It is also
cellular changes like membrane blebbing, mitochondrial
a potent chemopreventive agent inhibiting tumor promotion
membrane potential alteration, fragmentation of DNA into
against skin, oral, intestinal and colon carcinogenesis [9-11].
oligonucleosomal fragments and activation of cysteine
Many studies using curcumin focused on its
proteases called caspases finally culminating in cell death
antiinflammatory, antioxidant, anticarcinogenic, antiviral,
[16]. Curcumin has been shown to induce apoptosis in a
and antiinfective activities. In addition, the wound healing
variety of cells by several workers in the last decade, but no
and detoxifying properties of curcumin have also received
comprehensive review focused on the antiproliferative and
considerable attention [12]. Over the last decade, remarkable
apoptosis-inducing activities of curcumin is available so far
advances in our understanding of cancer biology have led us
although others have reviewed various biological activities
to the realization that apoptosis and the genes that control it
of curcumin [8, 12, 17-20]. Much progress has been made in
have a profound effect on the malignant phenotype although
understanding the molecular mechanism of apoptosis
apoptosis or programmed cell death is primarily a cell
induced by curcumin in cancer cells and its safety records are
suicide program critical for development, tissue
conducive for its therapeutic use at the preclinical/clinical
homeostasis, and protection against pathogens. It is now
stage for various cancers. At the same time, it is apparent
known that some oncogenic mutations disrupt apoptosis,
that most of the currently available drugs are not effective
leading to tumor initiation, progression or metastasis. In
against a subset of solid tumors because of the emergence of
contrast, other oncogenic changes during multistage
acquired chemoresistance. It will be quite interesting to see
whether the new experimental drugs like curcumin can be
*Address correspondence to this author at the Cancer Biology Laboratory, directed against such cancers with necessary interventional
Rajiv Gandhi Centre for Biotechnology, Thiruvananthapuram, Kerala 695 approaches. Our recent insight into how chemoresistance
014 India; Tel: 91-471-2347975; Fax: 91-471-2348096; E-mail: develops and the identification of new players in acquired
dkarunagaran@hotmail.com drug resistance offer great potential to design appropriate

1568-0096/05 $50.00+.00 © 2005 Bentham Science Publishers Ltd.

118 Current Cancer Drug Targets, 2005, Vol. 5, No. 2
interventional strategies to render curcumin-based therapy
more effective. The review places emphasis on novel
apoptosis-based potential targets that can be exploited to
sensitize drug-resistant cancers with this drug.
CHEMICAL STRUCTURE AND METABOLOITES
OF CURCUMIN
Commercial grade curcumin (77% curcumin, 17%
demethoxycurcumin and 3% bisdemethoxycurcumin)
contains pure curcumin shown in Fig. (1) with a molecular
weight of 368.37 and demethoxycurcumin, which are
equally potent as inhibitors of phorbol ester-induced tumor
promotion in mouse skin whereas bisdemethoxycurcumin is
relatively less active [21]. Curcumin is insoluble in water
but soluble in ethanol, dimethylsulfoxide and other organic
solvents. When curcumin was incubated in 0.1M phosphate
buffer and serum-free medium, pH 7.2 at 370C, about 90%
decomposed within 30 min. However, in cell culture
medium containing 10% fetal calf serum about 50% of
curcumin still remained stable after 8h. Trans-6- (4'-hydroxy-
3'-methoxyphenyl)-2,4-dioxo-5-hexenal was predicted to be
the major degradation product; vanillin, ferulic acid and
feruloyl methane were identified as minor degradation
products and the amount of vanillin increased with
incubation time [22]. Curcumin has poor bioavailability in
animals and humans due to its poor absorption and rapid
metabolism in the liver and intestinal wall [23-26]. The
major metabolites in suspensions of human and rat
hepatocytes were identified as hexahydrocurcumin and
hexahydrocurcuminol [25].
O O
H3CO OCH3
HO OH
Fig. (1). Chemical structure of curcumin
ANTIPROLIFERATIVE ACTIONS OF CURCUMIN
Cell cycle regulatory proteins and checkpoints are
downstream elements of cellular signaling cascades crucial
for cell proliferation. Curcumin inhibits the proliferation of a
wide variety of cancer cells derived from breast, blood,
colon, liver, kidney, prostate and skin through its effects on
different phases of cell cycle (Table 1). Human acute
myeloid leukemia cells (HL-60) are highly susceptible to
curcumin-mediated inhibition of cell proliferation in a dose-
and time-dependent manner leading to typical apoptosis. In
HL-60 cells treated with 25 µM curcumin for 48 h, 60.71±
1.20% cells were arrested in G2 /M phase of cell cycle at
first, then in G0 /G 1 phase and DNA synthesis was halted
resulting in apoptosis instead of differentiation [27] with
estimated IC50 values of 19.5 µM [42]. In HT-29 (human
colon cancer) cells curcumin causes cell cycle arrest in G2/M
phase but not apoptosis [33] whereas another study reported
induction of apoptosis by curcumin in these cells [43].
Curcumin inhibits cell proliferation in gastric (KATO-III)
and colon (HCT-116) cancer cells and induces G2/M arrest in
HCT-116 cells and cyclin B level remains unaffected,
whereas cyclin D and E levels decline with curcumin in both
cell lines [32]. Curcumin inhibits DNA synthesis in human
Karunagaran et al.
umbilical vein endothelial cells (HUVEC) as revealed by
[3H]-thymidine incorporation in a dose-dependent manner
without significantly affecting the viability of the cells.
Addition of curcumin to HUVEC resulted in an
accumulation of > 46% of the cells in early S-phase, as
determined by flow cytometric analysis. Curcumin caused a
significant loss of thymidine kinase activity, which may be
one of the possible mechanism(s) for the inhibition of DNA
synthesis activity of HUVEC by curcumin [36].
In response to lower concentrations of curcumin, mouse
embryonal carcinoma PCC4 cells ceased to proliferate and
showed cell cycle arrest at G1 phase after 4 hours of
treatment, followed by their differentiation, which is
characterized by an increase in nuclear/cytoplasmic ratio [39].
Curcumin inhibits the proliferation of head and neck
squamous cell carcinoma MDA 686LN cells by arresting the
cell cycle in G1 /S phase [30]. In vascular smooth-muscle
cells, this compound causes a G0/G1 cell-cycle arrest [38].
Gautam et al. investigated the antiproliferative effects of
curcumin against a variety of transformed and
nontransformed cell types. At equimolar concentrations
ranging from 6.25 to 50 µM, curcumin inhibited DNA
synthesis in five leukemia lines, three nontransformed
hematopoietic progenitor cell populations, and four
nontransformed fibroblastic cell lines in a concentration-
dependent manner. Curcumin also inhibited the cellular
growth of both transformed and nontransformed cells in
clonogenic assays. Without discriminating between
transformed or nontransformed cells, the inhibition of cell
proliferation by curcumin was not always associated with
programmed cell death [44]. Similarly inhibition of cell
proliferation by curcumin in many breast cancer cells was
not associated with apoptosis [41, 45]. Khar et al reported
that curcumin had no effect on the growth of nontransformed
cell lines, which showed neither superoxide generation nor
the induction of a stress response [46]. Interestingly,
curcumin had no growth-inhibitory effect on normal rat
hepatocytes and normal lung fibroblasts [47-49]. Similarly,
curcumin did not inhibit growth in primary culture of mouse
embryonic fibroblast C3H 10T1/2, rat embryonic fibroblast,
and human foreskin fibroblast cells in a concentration- and
time-dependent manner [43].
The mechanisms by which many cell types are arrested in
the G2/M-phase of the cell cycle upon curcumin treatment
are not known. MCF-7 breast cancer cells treated with 10-20
µM curcumin for 24-48 h are arrested in M-phase, and DNA
synthesis is almost completely inhibited, and the arrested
mitotic cells exhibit monopolar spindles, and chromosomes
do not undergo normal anaphase movements. After 48 h,
most cells eventually leave M-phase, and many form
multiple micronuclei instead of individual daughter nuclei.
These observations indicate that curcumin induces the
assembly of aberrant, monopolar mitotic spindles that are
impaired in their ability to segregate chromosomes. The
production of cells with extensive micronucleation after
curcumin treatment suggests that at least some of the
cytostatic effects of this phytochemical are due to its ability
to disrupt normal mitosis, and raises the possibility that
curcumin may promote genetic instability under some
circumstances [50]. Antiproliferative effects of curcumin thus
appear to be cell-type dependent and presumably the
experimental conditions and/or designs employed by
Implications for Cancer Therapy Current Cancer Drug Targets, 2005, Vol. 5, No. 2 119

Table 1. Effects of Curcumin on the Cell Cycle

Cell type Arrested cell cycle phase Cell cycle-related mechanisms References

HL-60 (Human acute myeloid G2/M first, then in G0/G1 Inhibition of DNA synthesis [27]
leukemia) phase

Human multiple myeloma cells G1/S Down regulation of cyclin D1, inhibition of IKK and NF-κB [28]

CA46 cells (human Burkitt's G0/G1 or G2/M and S Inhibition of DNA synthesis [29]
lymphoma)

MDA 686LN (human head and neck G1/S Down regulation of cyclin D1, inhibition of IKK and NF-κB [30]
squamous cell carcinoma)

HCT-116 (human colon cancer) G2/M P53-and p21-independent [31]

Down regulation of cyclin D and E but not B [32]
Activation of cdc2

HT-29 and HCT-15 (human colon G2/M Prostaglandin independent [33]

adenocarcinoma)

COLO205 (colorectal carcinoma) G1 Inhibition of Ca2+ dependent endonuclease, reduction of p53 [34]
gene expression

Lovo (colon cancer) S, G2/M inhibition of DNA synthesis [35]

HUVEC (human umbilical vein S inhibition of DNA synthesis [36]

endothelial cells)

ECV304 (immortalized human G0/G1 and/or G2/M up-regulation of p21WAF1/CIP1, p27KIP1, and p53 [37]
umbilical vein endothelial cells)

A7r5 (Rat aortic smooth muscle cell G0/G1 and S inhibition of protein tyrosine kinase activity, protein kinase C [38]
line) activity, c-myc mRNA expression and bcl-2 mRNA expression

PCC4 (mouse embryonal carcinoma G1 differentiation characterized by increase of [39]

cells) nuclear/cytoplasmic ratio

MCF-7/TH (multidrug-resistant human G2/M and subG0/G1 Reduction in the expression of Ki67, PCNA and p53 mRNAs [40]
breast carcinoma)

Human breast cancer cells S and G2/M inhibition of ornithine decarboxylase activity [41]

different researchers also contribute to these differences at
least, in part. Questions on the stability of curcumin
affecting its availability, formation of metabolites and their
relative contribution for the observed effects of curcumin
largely remain unanswered.
MECHANISMS OF CURCUMIN-INDUCED
APOPTOSIS
Curcumin is known to induce apoptosis in numerous
animal and human cell lines established from malignancies
like leukemia, melanoma, breast, lung, prostate, colon,
renal, hepatocellular and ovarian carcinomas (Table 2).
Curcumin also induces typical characteristics of apoptosis
like cell shrinkage, chromatin condensation, and DNA
fragmentation in immortalized mouse embryo fibroblast NIH
3T3 cells in a concentration- and time-dependent manner
[43]. Two distinct apoptotic signaling pathways have been
characterized in mammalian cells. One of these pathways
often called extrinsic or death receptor pathway involves
cytosolic domains of members of the TNFR (tumor necrosis
factor receptor) family such as CD 95/Fas and TRAIL-R1
(TNF-related apoptosis inducing ligand-R1) or TRAIL-R2,
which recruit procaspase 8. Ligation of these receptors leads
to oligomerization, recruitment of cytosolic adaptor FADD
(Fas associated death domain) and proteolytic activation of
procaspase 8, which then induces the activation of
downstream effector caspases like caspase 3, 6 and 7 [13,
51]. In the intrinsic or mitochondrial pathway, cytochrome c
is released from mitochondria into the cytosol resulting in
its high affinity interaction with apoptotic protease
activating factor-1 (Apaf-1) and subsequent activation of
caspase 9 [52]. The active caspase 9 then activates
downstream effector caspases that mediate the execution
phase exhibiting characteristic features of apoptosis, many of
which reflect the proteolytic cleavage of various intracellular
proteins ensuring peaceful elimination of the cell [53-55].
Apart from cytochrome c, the release of second mitochondria
derived activator of caspase (Smac) from mitochondria also
ensures continued caspase activation [56]. Internucleosomal
degradation, a characteristic biochemical feature of apoptosis,
results from the cleavage of ICAD (inhibitor of caspase
activated DNase or DNA fragmentation factor45/DFF45),
which releases an endonuclease called CAD (caspase
activated DNase).
Potential implications in chemoresistance mechanisms
make it highly relevant to ask the question of whether
curcumin induces apoptosis through extrinsic or intrinsic
apoptotic pathways specifically in different cancer cells.
120 Current Cancer Drug Targets, 2005, Vol. 5, No. 2 Karunagaran et al.

Table 2. Apoptotic Mechanisms Activated by Curcumin

Cell lines Apoptotic changes induced by Reference Cell lines Apoptotic changes induced Reference
curcumin by curcumin
DNA fragmentation [57] JNK-dependent apoptosis [62]
HL-60 (acute Caspase-3 activation PARP cleavage
myeloid leukemic DNA fragmentation [58] Human colon Activation of casapse-3 and [32]
cell line, human) Cytochrome c release cancer cell caspase-8
Activation of caspase-3 but not [59] lines Down regulation of Bcl-XL [63,64]
caspase-8 Nuclear condensation
Chromatin condensation DNA [60] Release of cytochrome c, [31]
fragmentation AIF and Smac
Down regulation of Mcl-1 [61] Activation of caspase-3,
Up regulation of Bax and Bak caspase-8, caspase-9
Up regulation of p27/kipl, p21/wafl Loss of mitochondrial
and pRbp transmembrane potential
Down regulation of cyclin D3 Cleavage of PARP and
Activation of caspase-8 and caspase- DFF45
3 p53- and p21-independent
PARP cleavage apoptosis
Bid cleavage Impairment of Wnt signaling
Cox-2 inhibition and cell-cell adhesion
Cytochrome c release pathways
Increase in p53 and Bax proteins and [65] Downregulation of NF-κB
Breast cancer cells p53DNA-binding activity Melanoma activation, iNOS and DNA- [68]
(human) Induction of redox signaling [66] (human) dependent protein kinase
Inhibition of H-ras-induced invasive catalytic subunit expression [69]
phenotype [67] Up regulation of p53, p21
Downregulation of Bcl-2 and MMP-2 (CIP1), p27 (Kip1) and
Upregulation of Bax checkpoint kinase 2
Inhibition of telomerase Activation of caspases-3 and
-8 but not caspase-9
Fas receptor aggregation in a
FasL-independent manner
Downregulation of NF-κB
activation and XIAP
expression
p53-dependent apoptosis Down regulation of of egr-1,
Human basal cell DNA fragmentation [70] BKS-2 c-myc, bcl-XL and p53 [71]
carcinoma Up regulation of nuclear p53 protein, (immature B expression
p21 (CIP1/WAF1) and Gadd45 cell
lymphoma)
Stage 4 MYCN- Up-regulation of p53, p21(WAF-
amplified 1/CIP-1) and Bax expression [72] K562 (human Activation of caspases-8 and [73]
neuroblastoma cell leukemia) 9
lines Inhibition of glutathione S-
transferase P1-1 expression
Generation of reactive oxygen
AK-5 (rat intermediates [74,75] MDA 686LN IKK and NF-κB inhibition [30]
histiocytoma) Activation of caspase-3 (human head Reduced expression of Bcl-2,
Loss of mitochondrial membrane and neck cyclin D1, IL-6, COX-2 and
potential squamous cell MMP-9
Cytochrome c release carcinoma)
DNA fragmentation Increased caspase-3
A7r5 (Rat aortic Inhibition of protein tyrosine kinase [38] Vγ9vδ2 T cells expression in nucleus [76]
smooth muscle cell and PKC activities and c-myc and PARP cleavage
line) bcl-2 mRNA expression. AIF translocation to nucleus
Activation of caspase 3
CA46 cells (human DNA fragmentation Decreased [29] Human renal Cleavage of phospholipase [77]
Burkitt's lymphoma) protein and mRNA expression of c- Caki cells C-gamma1
myc, bcl-2, mutant-type p53 Release of cytochrome c
Increased Fas protein and mRNA DNA fragmentation
Dephosphorylation of Akt
Down-regulation of Bcl-2,
Bcl-XL and IAP proteins
Up-regulating Caspase-3 and down- p53-independent mechanism
A2780 (human regulating expression of NF-κB [78] Human lung Decrease in expression of [48]
ovarian cancer) cancer cells p53, bcl-2, and bcl-XL

DNA fragmentation Down regulation of the

Ehrilich’s Ascites Cytochrome c release Activation [79] Human expression of Bcl-2 and Bcl- [80]
Carcinoma cells of caspase 3 prostate xL
Up regulation of Bax cancer cells Activation of caspase-3 and
caspase-8
Implications for Cancer Therapy
Curcumin appears to induce apoptosis in human melanoma
cells through a Fas receptor/caspase-8 pathway and the
expression of dominant negative FADD significantly
inhibits curcumin-induced apoptosis [69]. In gastric (KATO-
III) and colon (HCT-116) cancer cells curcumin caused
cleavage of poly (ADP) ribose polymerase (PARP, a
substrate of caspase-3), activation of caspase-3 and caspase-8
initiating the Fas signaling pathway of apoptosis [32].
Induction of Fas ligand in a few tumor cells [46] and
increased expression of Fas protein and mRNA in Burkitt's
lymphoma cells occur upon treatment with curcumin [29].
To show conclusively that curcumin acts through Fas
signaling, synthesis of FasL should be evident before the
cells become apoptotic and cytotoxicity should be inhibited
by manipulations that block FasL-Fas and FADD-procaspase
8 interactions and cells that lack Fas should fail to respond
to the drug. Unfortunately, such rigorous evidences are
mostly lacking on the involvement of Fas signaling in
response to curcumin.
Curcumin induces membrane permeability, swelling,
loss of membrane potential and inhibition of ATP synthesis
mediated by the opening of the permeability transition pore
in isolated rat liver mitochondria [81]. Activation of caspase
3 and cleavage of PARP and Bid, release of mitochondrial
cytochrome c and activation of caspase 8 (blocked by
dominant negative FLICE but not by dominant negative
FADD) in response to curcumin (25µM) are in favor of
mitochondrial pathway for curcumin-induced apoptosis of
HL-60 cells [61]. Surprisingly another report found no
activation of caspase 8 in the presence of curcumin (50 µM)
in HL-60 cells [58]. It is not clear whether this striking
difference is due to the different concentrations of curcumin
and/or other experimental conditions used by these
researchers. From the available data (Table 2) it appears that
curcumin-induces apoptosis of many tumor cells mainly by
the mitochondrial pathway and more studies are needed to
implicate or rule out a role for the extrinsic pathway in
curcumin-induced apoptosis. A mitochondrial flavoprotein
called apoptosis-inducing factor (AIF) translocates to the
nucleus when apoptosis is induced and recombinant AIF
causes chromatin condensation in isolated nuclei and large-
scale fragmentation of DNA independent of caspase
activation [82]. Similarly a mitochondrion-specific nuclease
known as endonuclease G translocates to the nucleus during
apoptosis and cleaves chromatin DNA into nucleosomal
fragments independently of caspases [83]. There is paucity of
information on the role of curcumin in regulating the release
of caspase-independent proapoptogenic factors such as AIF
and endonuclease G although a few laboratories including
ours have shown the release of AIF in response to curcumin
[63, 64, 76, 84]. It is hoped that these gaps will be filled
soon and as the biochemical and molecular complexities of
the apoptotic pathways are elucidated, new molecular targets
for curcumin will be identified.
Alteration of p53 (tumor suppressor) is the most
common mutation in human cancer, and it functions as a
transcription factor regulating genes (p53 can up regulate
Bax and Fas) involved in cell cycle arrest, DNA repair and
apoptosis. Curcumin treatment causes p53- and p21-
independent cell cycle arrest and apoptosis in p53 (+/+), p53
(-/-) and p21 (-/-) derivatives of HCT-116 human colon
cancer cells [31]. In addition, curcumin induces apoptosis in
Current Cancer Drug Targets, 2005, Vol. 5, No. 2 121
both p53 proficient (A549) and p53 null mutant (H1299)
lung cancer cell lines with a decrease in expression of p53
suggesting a p53-independent induction of apoptosis in lung
cancer cells [48]. Curcumin treatment causes a reduction in
the expression of Ki67, PCNA, and p53 mRNAs in MCF-
7/TH, a multidrug-resistant human breast cancer cell line
[40]. However, curcumin also induces nuclear p53 protein
and its downstream targets, including p21 (CIP1/WAF1)
and Gadd45 in human basal cell carcinoma cells [70].
Increase in p53 as well as its DNA-binding activity occurs
during curcumin-induced apoptosis of MCF-7 (human breast
cancer) cells [65]. Experiments using p53-null MDAH041
cells as well as low and high p53-expressing TR9-7 cells
also support a p53-dependent pathway for curcumin induced
apoptosis in tumor cells [65]. These data suggest that the
p53-associated signaling pathway is differentially regulated
by curcumin in a cell type-dependent manner to induce
apoptosis.
Oxidative stress signals induced by the formation of
reactive oxygen species (ROS) and glutathione depletion are
also considered as important activators of apoptosis [13].
Being lipophilic, curcumin can collapse transmembrane
mitochondrial potential and increase permeability for proton
and interfere with energy the coupling system in the
mitochondria. As a consequence of energy dissipation in
mitochondria, cells are likely to suffer from increased levels
of ROS and, sensitivity of many tumor cells to curcumin
correlates with the generation of superoxide radicals [46].
Depletion of glutathione by buthionine sulfoximine resulted
in the increased generation of ROS, thereby further
sensitizing the tumor cells to curcumin [47]. In contrast,
curcumin is a potent scavenger of a variety of ROS
including superoxide anion, hydroxyl radical, singlet
oxygen, nitric oxide and peroxynitrite [85, 86]. Curcumin
also enhances the activities of antioxidant enzymes [87] and
increases glutathione levels [88], which is expected to
protect cells by preventing thiol depletion occurring during
apoptosis. In addition, curcumin exhibits both pro- and
antioxidant activities and the prooxidant DNA cleavage
activity is the consequence of binding of Cu (II) to various
sites on the curcumin molecule [89]. Other well-known
antioxidants such as N-acetyl L-cysteine, L-ascorbic acid,
alpha-tocopherol, catalase and superoxide dismutase have all
prevented curcumin-induced apoptosis effectively in HL-60
cells [90]. Due to the complexity of redox regulation by
curcumin as described above, it is very difficult to provide a
mechanistic basis for the induction of apoptosis by curcumin
by this mechanism at the moment.
Curcumin (50 µM) induces apoptosis-like cell death in
quiescent rat thymocyte and splenocytes, human peripheral
blood lymphocytes, proliferating IL-2-dependent T-cells,
human leukemic (MOLT-4) cells and mouse leukemic
(L1210) cells, and only in HL-60 cells classical apoptosis
occurs [57]. Similarly curcumin-induced changes in nuclear
morphology, DNA fragmentation, the extent and time-course
of membrane dynamics are different from typical apoptosis
induced by dexamethasone in rat thymocytes [91].
Apoptosis induced by curcumin in Jurkat cells also differs
from "classical apoptosis" by the lack of mitochondrial
depolarization and of the involvement of caspases [92, 93].
Jaruga et al. reported that curcumin expands human
erythrocyte membrane inducing echinocytosis accompanied
122 Current Cancer Drug Targets, 2005, Vol. 5, No. 2
by transient exposure of phosphatidylserine and lipid
fluidity changes [94]. These authors caution researchers that
such nonspecific apoptosis-independent effects of curcumin
on cellular membranes would produce artifacts of apoptosis
measurement, since several methods are based on membrane
changes.
In contrast to the apoptosis-inducing effects described
above, dietary curcumin can inhibit chemotherapy-induced
apoptosis through inhibition of ROS generation and
blockade of JNK function [95]. Curcumin also inhibited
camptothecin-, mechlorethamine-, and doxorubicin-induced
apoptosis of MCF-7, MDA-MB-231, and BT-474 human
breast cancer cells by up to 70%. Inhibition of programmed
cell death was time and concentration dependent, but
occurred after relatively brief 3-h exposures, or at curcumin
concentrations of 1 µM [95]. Curcumin inhibited apoptosis
in dexamethasone-treated rat thymocytes accompanied by
partial suppression of AP-1 activity [96]. The capacity of
curcumin to inhibit both cell growth and death strongly
implies that these two biological processes share a common
pathway at some point and that curcumin affects a common
step, presumably involving a a glutathione-independent
mechanism of cell protection [97]. The cytoprotective effect
of curcumin against Shiga toxin-induced injury in cultured
human proximal tubule epithelial cells may be a
consequence of an increased expression of hsp70 [98].
It is not perfectly clear as to what cellular conditions are
contributing to the induction of apoptosis by curcumin as
opposed to the conditions in which curcumin is not able to
induce apoptosis and the conditions that direct it to protect
cells from apoptosis. Apoptosis-inducing activities of
curcumin derivatives in various cell types have not been
studied in detail. There is also a need for synthetic
derivatives of curcumin that induce apoptosis more
effectively with better bioavailability.
IMPLICATIONS FOR CANCER THERAPY
Basically, cancer therapy aims to kill the cancer cells by
apoptosis very selectively without affecting the normal cells
and in this context it is very important to understand the
mechanisms responsible for drug-induced apoptosis
especially to choose or improve strategies for treatment.
Tumor progression needs proteins that promote cell
proliferation but additionally requires the expression of
antiapoptotic proteins and/or the inactivation of proapoptotic
proteins. Although the molecular mechanisms of
antiproliferative and apoptotic effects of curcumin remain
elusive, studies on the expression of specific proteins that
regulate apoptosis in various cell types following treatment
with curcumin have provided several clues to formulate
strategies of cancer treatment with this compound.
Different steps in the two apoptotic pathways are highly
regulated and one of the earliest steps is the activation of
caspase 8 that can be inhibited by FLIPs (FADD-like
interleukin-1-β-converting enzyme like protease inhibitory
proteins) [13]. Multi-domain Bcl-2 family of proteins are
important regulators of apoptosis that can be grouped into
the antiapoptotic members comprising Bcl-2, Bcl-XL and
Mcl-1, the proapoptotic members such as Bax and Bak and
the BH3 domain only proteins Bim, Bid and Bik [99]. Bcl-
Karunagaran et al.
2 and Bcl-XL perform their antiapoptotic function by
inhibiting the release of cytochrome c from mitochondria by
preventing the translocation or activation of Bax or Bak
[100, 101]. Bid is a substrate for caspase 8, and after the
proteolytic cleavage, it gets truncated to form tBid, which
translocates to mitochondria to oligomerize Bak or Bax
promoting the release of cytochrome c [100, 102]. This
connects the death receptor and mitochondrial apoptotic
pathways to amplify the signals downstream of caspase
activation. Apart from Bid-mediated connection between the
two apoptotic pathways, additional cross talk can also occur
such as the caspase 6-mediated activation of procaspase 8
[55], but no information on this is available for curcumin-
induced apoptosis.
Tumor cells proliferate by overexpressing oncogenes such
as c-myc, and curcumin down regulates the expression of
survival genes c-myc, egr-1, and bcl-XL in B cell lymphoma
cells [71]. Curcumin induces apoptosis in lung and prostate
cancer cell lines with a decrease in expression of Bcl-2, and
Bcl-XL [48, 80]. In smooth muscle cells, significant
reduction in Bcl-2 m RNA was detected with curcumin [38].
In HL-60 cells, Bcl2 protein level decreased to 30% after 6 h
of curcumin treatment and a further 6 h treatment
subsequently reduced it to 20% [90]. Overexpression of Bcl-
2 in HL-60 cells resulted in a delay of curcumin-treated cells
entering into apoptosis [90]. Similarly, HL-60 cells made to
overexpress Bcl-2 or Bcl-XL completely blocked curcumin-
mediated induction of cytochrome c release, caspases 8 and
3, and cleavage of Bid and PARP [61]. However, Bcl-2 over
expression fails to protect AK-5 cells against curcumin
induced apoptosis [103]. Curcumin up regulates Bax but
Bcl-2 remains unchanged in Ehrlich’s Ascites carcinoma
cells, bypassing the Bcl-2 checkpoint and overriding its
protective effect on apoptosis [79]. Similarly, the apoptosis
suppressor Bcl-2 and promoter Bax were not changed with
curcumin treatment in basal cell carcinoma [70]. In non-
apoptotic cells, Bax is a soluble monomeric protein
diffusely distributed in the cytoplasm [104]. Bax deficiency
renders cancer cells resistant to several anticancer drugs
acting via the mitochondria or endoplasmic reticulum stress
[105, 106]. Using Bax+/- and Bax-/- human colon cancer
cells, we have found an essential role for Bax in curcumin-
induced apoptosis (unpublished observations).
Ku 70, a subunit of Ku protein complex involved in
DNA repair, plays a major role in keeping the Bax protein in
inactive conformation even during apoptotic stresses [107].
High expression of Ku70 observed in cancer cells suggests
additional roles for this protein in cancer progression and
chemoresistance [108, 109]. Our laboratory has shown that
Bcl-XL and Ku70 protect human colon cancer cells by
inhibiting the release of cytochrome c, Smac and AIF, and
the activation of caspases 9, 8 and 3 triggered by curcumin
[84]. Cells transfected with antisense Bcl-XL or Ku70 were
more sensitive to curcumin through enhanced activation of
caspases 9 and 3 and release of cytochrome c, Smac and
AIF. Curcumin-induced activation of caspase 8 was blocked
by Ku70 but not by Bcl-XL. Cells made to overexpress Bax
were highly sensitized to curcumin when Ku70 was down
regulated [84].
Inhibitors of apoptosis proteins (IAPs) such as XIAP, c-
IAP1, c-IAP2 and survivin, are another class of regulatory
Implications for Cancer Therapy
proteins that can bind and inhibit caspases, but IAPs can be
inhibited by the binding of Smac promoting caspase
activation [13]. Bush et al. reported the suppression of
XIAP in human melanoma cells in response to curcumin
treatment [69]. Transfection of extrinsic Smac gene results in
its over-expression in gastric cancer (MKN-45) cells, which
sensitized these cells to curcumin-induced apoptosis [110].
Recent experimental evidence suggests that heat shock
proteins like hsp70, 90 and 27 can also function as
antiapoptotic proteins by suppressing the signaling events of
apoptosis [111-113]. High expression of Hsps especially
Hsp27 and Hsp70, in breast, endometrial, or gastric cancer
has been associated with metastasis, poor prognosis and
Fig. (2). Known regulators of curcumin-induced apoptosis
Current Cancer Drug Targets, 2005, Vol. 5, No. 2 123
resistance to chemo- or radio- therapy [112]. Curcumin-
resistant tumor cell lines showed significantly higher
production of Hsp70, while the sensitive cells produced only
basal levels of Hsps [46]. Our laboratory results show that
heat shock partly prevents the curcumin-mediated release of
AIF but not that of Smac and cytochrome c from
mitochondria and reduces the activation of caspases 9 and 3
but not 8 induced by curcumin in human colon cancer cells
[63]. These cells were protected from curcumin-induced cell
death by Hsp70 while cells harboring antisense Hsp70
(Ashsp70) were highly sensitive to curcumin. Loss of
mitochondrial transmembrane potential induced by curcumin
was further accelerated by antisense Hsp70 expression and
124 Current Cancer Drug Targets, 2005, Vol. 5, No. 2
Hsp70 restored it partly. Ashsp70 cells released more
cytochrome c, AIF and Smac from mitochondria upon
curcumin treatment than control cells. Hsp70 partly
prevented the release of AIF but not the other proteins.
Activation of caspases 3 and 9 induced by curcumin was
also inhibited by Hsp70, whereas more activation could be
seen in Ashsp70 cells, although caspase 8 activation was
unaffected by changes in Hsp70 expression. Curcumin-
induced cleavage of PARP and DFF45 was inhibited by
Hsp70 but enhanced in Ashsp70 cells [64]. Our studies
demonstrated the potential of Hsp70 in protecting SW480
cells from curcumin-induced apoptosis and highlighted that
silencing the expression of Hsp70 is an effective approach to
augment curcumin-based therapy in cancers that are resistant
due to Hsp70 expression. It is apparent that many molecules
regulate curcumin-induced apoptosis by interfering with the
mitochondrial pathway as shown in Fig. (2).
Transcription factors such as AP-1 and a number of other
signal transduction intermediators including adhesion
molecules, several kinases such as receptor tyrosine kinases,
mitogen activated protein kinases (MAPKs) and c-jun N-
terminal kinases (JNKs) influence the course of drug-induced
apoptosis. Short-term treatment of human epidermoid
carcinoma (A431) cells with curcumin inhibited EGF
receptor intrinsic kinase activity up to 90% in a dose- and
time-dependent manner, and also inhibited EGF-induced
tyrosine phosphorylation of EGF receptors. The observed
early effects of curcumin were mediated via a cellular
mechanism(s), and preceded the period when inhibition of
cell growth occurred [114]. Curcumin effectively inhibited
p185neu tyrosine kinase receptor activity by depleting
p185neu protein and potently suppressed the growth of
multiple breast cancer cell lines [45].
Curcumin increases growth arrest and DNA damage-
inducible gene 153 (GADD153) mRNA (and also protein)
expression before the appearance of apoptotic features
without depending on upstream MAPK [115]. It appears that
JNK, but not p38 or extracellular signal-regulated protein
kinase signaling, plays an important role in curcumin
mediated apoptosis in human colon cancer cells [62].
Many cancer cells protect themselves against apoptosis
by activating nuclear factor-kappaB (NF-κ B/Rel), a
transcription factor that promotes cell survival [116].
Curcumin induces apoptosis by down-regulating NF-κB
expression in ovarian cancer cells [78] or its activity in
melanoma cells [68]. To understand the role of NF-κB in
curcumin-induced apoptosis, we stably transfected relA gene
encoding the p65/RelA subunit of NF-κB, into L-929
(mouse fibrosarcoma) cells and the relA-transfected cells
were resistant to varying doses of curcumin, whereas the
parental cells underwent apoptosis in a time- and dose-
dependent manner [117]. Recently, it has been reported that
fresh CD138+ cells derived from multiple myeloma patients
express constitutively active NF-κB and STAT3, and
suppression of these transcription factors with curcumin
inhibits the survival of the cells leading to apoptosis [118].
Akt, a serine/threonine protein kinase and a downstream
target of phosphatidyl inositol 3-kinase, suppresses
apoptosis by activating NF-κB [119, 120]. One of the
mechanisms of curcumin’s action may be via inhibition of
Akt as it completely inhibited Akt activation in LNCaP and
Karunagaran et al.
PC-3 cells in which Akt is constitutively active [121].
Blocking peroxisome proliferator-activated receptor-gamma
(PPAR-γ) activation abrogated the effects of curcumin on
induction of apoptosis and inhibition of the expression of
extracellular matrix (ECM) genes in activated hepatic stellate
cells (HSC) in vitro [122]. Furthermore, curcumin
suppresses the gene expression of TGF-β receptors and
interrupts the TGF-β signaling pathway in activated HSC,
mediated by PPAR-γ activation [122]. These results
demonstrated that curcumin stimulated PPAR-γ activity in
activated HSC in vitro, which is required for curcumin to
reduce cell proliferation, induce apoptosis and suppress
ECM gene expression.
Curcumin has the ability to inhibit iNOS induction by
LPS in the mammary gland and to scavenge NO radicals
[123]. In ex vivo cultured BALB/c mouse peritoneal
macrophages, 1-20 µM of curcumin reduced the production
of iNOS mRNA in a concentration-dependent manner [124].
The length of telomeres at chromosome ends shortens
during each cell cycle, but after reaching a critical shortening
the sensors of DNA damage start inducing p53-dependent
cell cycle arrest or apoptosis [125]. However, in tumor cells,
increased telomerase activity takes care of teleomere
stabilization [126]. Curcumin inhibits telomerase activity in
human breast cancer cells (MCF-7) by down regulating the
expression of human telomerase reverse transcritpase [67].
The growth inhibitory effect of curcumin in breast cancer
cells was time- and dose-dependent, and correlated with its
inhibition of ornithine decarboxylase activity [41].
Curcumin is also known to inhibit PKC [127],
cyclooxygenase and lipoxygenase enzymes [128].
Curcumin enhances the cytotoxicity of chemotherapeutic
agents in prostate cancer cells by inducing p21
(WAF1/CIP1) and C/EBPbeta expression, and suppressing
NF-κB activation [129]. Curcumin enhances the sensitivity
of prostate tumor cells to TRAIL by inhibiting NF-κB
activation through blockade of phosphorylation of IκBα and
its degradation [130, 131]. Curcumin increases the
sensitivity of ovarian cancer cells (CAOV3 and SKOV3) to
cisplatin [132]. Combining curcumin with 5-fluorouracil
significantly increased growth inhibition of AGS human
gastric carcinoma cells compared with either curcumin or 5-
FU alone suggesting synergistic actions of the two drugs
[133].
Aggarwal has recently patented his findings showing that
a combination of curcumin and paclitaxel (taxol) can be used
to treat and inhibit metastasis of breast tumors
(US2004002499). When tumor cells were pretreated with
common biologic modulators such as tamoxifen,
dexamethasone, and curcumin, the doxorubicin-induced NF-
κB activation was attenuated significantly [134]. Vinblastine
sensitivity to multidrug-resistant cervical cells KB-V1 was
increased by curcumin [135]. Curcumin-induced down-
regulation of NF-κB also induced chemosensitivity to
vincristine and melphalan in human multiple myeloma cells
[28].
Synergistic inhibitory effects of 13-cis retinoic acid and
curcumin have been reported on the growth of head and neck
squamous cell carcinoma cells [136]. Combinations of
curcumin and retinoic acid have a particularly potent
Implications for Cancer Therapy
Fig. (3). Potential strategies targeting curcumin-induced apoptotic pathway to overcome chemoresistance in cancers
inhibitory effect on the proliferation of HL-60 cells [137].
Down-regulation of Bcl-2 in curcumin plus radiation-treated
PC-3 cells, together, altered the Bcl-2/Bax ratio and this
caused the enhanced radiosensitization effect [138]. Piperine
enhances the serum concentration, extent of absorption and
bioavailability of curcumin in both rats and humans [26] and
the biological effects of such a potentially useful
combination need to be understood in the future. Based on
our work and the available literature, we have outlined some
of the potential strategies that can be employed in curcumin-
based therapy in Fig. (3).
Studying the expression changes of thousands of genes at
a given time and/or condition may provide increased insight
into the mechanism of action of curcumin in cancer cells,
and increasing our understand of how this compound can
protect against carcinogenesis. Microarray analysis revealed
the down regulation of 81 genes and up regulation of 71
genes after curcumin treatment in highly invasive lung
adenocarcinoma cells. Below sublethal concentrations (10
µM), several invasion-related genes were suppressed and
several heat-shock proteins (Hsp27, Hsp70 and Hsp40-like
proteins) were induced by curcumin [139]. Curcumin was
found to up regulate cdk inhibitor genes, and slightly down
regulate cyclin B1 and cdc2 in human umbilical vein
endothelial cells [37]. Gene expression changes were studied
in human colon cancer cell lines, using cDNA microarrays
with four thousand human genes, and curcumin modulated a
Current Cancer Drug Targets, 2005, Vol. 5, No. 2 125
number of cell cycle genes and p53 [140]. Microarray studies
need to be extended to cell lines of different tissues
especially because curcumin is known to induce cell-type
dependent changes.
Inspite of many in vitro studies on curcumin-induced
apoptosis, studies conducted in animals and humans have
not focused on measuring apoptosis-related parameters.
Fifteen patients with advanced colorectal cancer refractory to
standard chemotherapies received curcuma extract daily for
up to 4 months. Oral curcumin extract was tolerated well
and dose-limiting toxicity was not observed [141].
Curcumin was also used without any notable side-effects or
toxicity in patients suffering from idiopathic inflammatory
orbital pseudotumors [142] and phase-II clinical trials were
conducted with curcumin in patients with urinary bladder
cancer, uterine cervical intraepithelial lesions, oral
leukoplasia and intestinal metaplasia of the stomach [143].
Among naturally occurring compounds, curcumin is fit to be
a front-runner in the race to the drug market as it is a
pharmacologically safe compound (when administered at
doses up to 10 g/day, human clinical trials indicated no
dose-limiting toxicity) [17,143].
CONCLUSIONS AND FUTURE DIRECTIONS
It is generally believed that chemotherapy eliminates pre
neoplastic or neoplastic cells by the induction of apoptosis,
126 Current Cancer Drug Targets, 2005, Vol. 5, No. 2
but the other possibility that some forms of chemotherapy-
induced cell death cannot be readily classified as apoptosis
or necrosis exists [144, 145]. Lysosomes have recently
emerged as the central players of apoptotic cell death in
addition to mitochondria and lysosomal proteases from the
cathepsin family frequently translocate from lysosomal
lumen to the cytosol to promote cell death. Cathepsin B and
Cathepsin D act as the main executors of caspase-
independent cell death induced by TNF-α and several
chemotherapeutic agents [146, 147]. With the present
knowledge it is not very clear whether curcumin being a
lipophilic compound known to induce apoptotic like
changes independent of caspases can amplify lysosomal
mediated cathepsin release. Novel apoptotic related proteins
in this and other unknown mechanisms regulated by
curcumin can be rapidly identified with a proteomic
approach and such studies will also provide a strong
rationale for the selection of therapeutic targets for
personalized treatment regimens. Presumably all the
potential approaches highlighted in this review may not
reach the clinic but serve only to demonstrate the proof of
concept, but it is high time that the knowledge gained is
translated into clinical applications. Time-tested
pharmacological safety, multiple molecular targets favorable
to treat a genetic disease like cancer with multiple defects,
scope for improving bioavailability and/or biological
activity (through synthetic curcuminoids of desired activity
aided by molecular modeling) vouch in favor of curcumin to
be developed as a drug for prevention and therapy of various
cancers either alone or in combination with standard drugs or
novel approaches.
ACKNOWLEDGEMENTS
Work in the authors’ laboratory was supported by
funding from the Kerala State Council for Science,
Technology and the Environment (to D. K.), a grant from
the Life Sciences Research Board, Defence Research
Development Organization, Government of India (to D. K.
and S. K.) and a Senior Research Fellowship (to R. R.) of
the Council of Scientific and Industrial Research,
Government of India. The authors thank Goodwin Jinesh for
technical help.
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