Vol. 53 No. 3/2006, 531–538
on-line at: www.actabp.pl
Curcumin induces cell death without oligonucleosomal DNA fragmen-
tation in quiescent and proliferating human CD8+ cells
Adriana Magalska, Agnieszka Brzezinska, Anna Bielak-Zmijewska,
Katarzyna Piwocka, Gra?yna Mosieniak and Ewa Sikora½
Laboratory of Molecular Bases of Aging, Nencki Institute of Experimental Biology, Warszawa, Poland;
Received: 30 June, 2006; revised: 01 August, 2006; accepted: 21 August, 2006
available on-line: 02 September, 2006
Cytotoxic CD8+ cells play an important role in determining host response to tumor, thus chemo-
therapy is potentially dangerous as it may lead to T cells depletion. The purpose of this study
was to elucidate the propensity of quiescent and proliferating human CD8+ cells to undergo cell
death upon treatment with curcumin, a natural dye in Phase I of clinical trials as a prospective
chemopreventive agent. Methods: We treated human quiescent or proliferating CD8+ cells with
50 ?M curcumin or irradiated them with UVC. Cell death symptoms such as decreased cell vi-
ability, chromatin condensation, activation of caspase-3 and specific DFF40/CAD endonuclease
and oligonucleosomal DNA fragmentation were analyzed using MTT test, microscopic observa-
tion, Western blotting and flow cytometry. Results: Curcumin decreased cell viability, activated
caspase-3 and decreased the level of DFF45/ICAD, the inhibitor of the DFF40/CAD endonuclease.
However, this did not lead to oligonucleosomal DNA degradation. In contrast, UVC-irradiated
proliferating, but not quiescent CD8+ cells revealed molecular and morphological changes charac-
teristic for apoptosis, including oligonucleosomal DNA fragmentation. Curcumin can induce cell
death in normal human lymphocytes both quiescent and proliferating, without oligonucleosomal
DNA degradation which is considered as a main hallmark of apoptotic cell death. Taking into
account the role of CD8+ cells in tumor response, their depletion during chemotherapy could be
Keywords: CD8+, cell death, curcumin, DNA degradation
& Shi, 2004). One of the hallmarks of the terminal
stages of apoptosis is oligonucleosomal DNA frag-
Apoptosis, or programmed cell death, is a mentation (Wyllie et al., 1980). Recent years have led
fundamental process essential for both development to the discovery of two major apoptotic nucleases,
and maintenance of tissue homeostasis (reviewed termed DNA fragmentation factor (DFF) or caspase-
in Jacobson et al., 1997). Cells undergoing apopto-
activated DNase (CAD) and endonuclease G (Endo
sis exhibit specific morphological changes includ-
G). In non-apoptotic cells, DFF exists in the nucleus
ing membrane blebbing, cytoplasmic and chromatin as a heterodimer, composed of a 45 kDa chaperone
condensation, DNA fragmentation, nuclear break-
and inhibitory subunit (DFF45 or ICAD-L) and a 40
down and assembly of membrane-enclosed vesicles kDa latent nuclease subunit (DFF40/CAD). Apopto-
termed apoptotic bodies, eventually subjected to tic activation of caspase-3 or -7 results in the cleav-
phagocytosis (reviewed in Wyllie et al., 1980). Mul-
age of DFF45/ICAD and release of the DFF40/CAD
tiple apoptotic stimuli trigger the activation of pro-
nuclease which then forms active homo-oligomers
teases called caspases, which in turn initiate and (reviewed in Widlak & Garrard, 2005).
execute the apoptotic program (reviewed in Riedl
Abbreviations: 7-AAD, 7-aminoactinomycin D; AICD, activation-induced cell death; APCs, antigen presenting cells; CAD,
casapase-activated DNase; CFSE, carboxyfluorescein diacetate succinimidyl ester; DFF, DNA fragmentation factor; Endo
G, endonuclease G; FCS, fetal calf serum; HA, host antigen; mAb, monoclonal antibody; MDR, multidrug resistance;
MTT, 3-(4,5-dimethyldiazol-2-yl)-2,5-diphenyl tetrazolium bromide; PBS, phosphate-buffered saline; PHA, phytohaemag-
glutinin; TCR, T-cell receptor.
Human lymphocytes undergo so called “ac-
min, and its many uses, has led to studies aimed at
tivation-induced cell death” (AICD) when activated elucidating some of its activities with particular at-
lymphocytes are induced to die during the down-
tention on the anticancer activity. These flourished
phase of their immune response. This is caused by with several Phase I human trials that have shown
ligation of the Fas receptor (CD95) with Fas ligand this compound to be well tolerated (Cheng et al.,
(CD95-L) whose expression is up regulated by TCR 2001; Hsu et al., 2002; Iqbal et al., 2003; Sharma et al.,
activation (Krammer, 2000). AICD can be mimicked 2004).
in vitro by stimulation of TCR with non-specific mi-
It has been shown that curcumin can inhibit
togen, phytohaemagglutinin (PHA), or by specific proliferation and/or induce cell death in in vitro
stimulation of TCR/CD28 with anti CD3/antiCD28. experiments with different cancer cells. The most
A second type of physiological cell death of T cells common cell death mode upon curcumin treatment
caused by deprivation of survival stimuli, such as seems to be apoptosis (reviewed in Lin et al., 2000;
cytokines, leading to down-regulation of anti-apop-
Karunagaran et al., 2005). There are also reports
totic proteins such as Bcl-2 can be called “passive showing that unlike in cancer cells, curcumin does
cell death” (Akbar & Salmon, 1997).
not induce apoptosis in normal cells (Jiang et al.,
Moreover, normal lymphocytes can under-
1996). However, we showed that curcumin induced
go cell death induced by drugs as a side-effect of cell death not only in cancer but also in normal rat
chemotherapy aimed at malignant cells. This type and human lymphocytes (Bielak-Zmijewska et al.,
of lymphocyte cell death can be included in the cat-
2000). The cell death induced with curcumin in nor-
egory of damage-induced cell death (De Martinis et mal and transformed lymphocytes was not charac-
al., 2005). Usually chemotherapy causes severe tox-
terized oligonucleosomal DNA fragmentation that is
icity in normal tissues, leading to side-effects such typical for apoptosis. Moreover, we showed in Jurkat
as mucosistis, hair loss, and myelosuppression. In cells that curcumin induced caspase-3 activation and
addition, chemotherapy induces acute lymphopenia following DFF40/CAD activation but concomitantly
and chronic depletion of T cells, leading to increased blocked the active centre of the endonuclease thus
susceptibility to opportunistic infections.
precluding DNA fragmentation (Sikora et al., 2006).
CD8+ cells play a very important role in the Here we investigated whether also in normal human
anti-tumor immune response. Antigens from pe-
quiescent and proliferating lymphocytes curcumin
ripheral tumor cells can enter the class I pathway gives the same symptoms, namely cell death with
for presentation by host antigen (HA) presenting activation of caspase-3 and DFF40/CAD endonucle-
cells (APCs) to CD8 cells, a process commonly ase but without oligonucleosomal DNA fragmenta-
known as ”cross-presentation” (Heath & Carbone, tion. As the oligonucleosomal DNA degradation is
2001). It has recently been shown in mice that not considered a main hallmark of apoptotic cell death
only the tumor presence but also induction of tu-
the lack of this sort of degradation can be improp-
mor cell apoptosis in vivo increases tumor antigen erly interpreted as an absence of apoptosis.
cross-presentation of HA to CD8 cells which are
not deleted but primed as was revealed by CFSE
proliferation assay (Nowak et al., 2003). Quantita-
MAteRIAls AnD MethoDs
tion of lymphocyte populations in the peripheral
blood of patients enrolled in multiagent dose-inten-
T-cell preparation and treatment. Mononu-
sive regiments revealed significant lymphocyte de-
clear cells were obtained by standard centrifugation
pletion, but with a more profound effect on CD4+ over Ficoll-Paque from lymphocyte buffy coat (about
than on CD8+ T-cells (Mackall, 2000). Accordingly, 108 cells) of peripheral blood of eight healthy blood
in this paper we checked interested whether CD8+ donors (aged 22–33 years). The buffy coats were ob-
cells, both quiescent and activated, might be indeed tained from the Regional Centre for Blood Donation
resistant to apoptosis induced by a classical DNA and Blood Treatment in Warsaw (Poland). All cell
damaging agent, UVC, and the natural dye curcu-
cultures were conducted in RPMI 1640 supplemented
with 10% FCS, 2 mM l-glutamine and antibiotics, at
Curcumin (diferuloyl methane) is a naturally a starting density of 0.8 × 106 cells/ml. The cells were
occurring yellow pigment derived from the rhizome stimulated by PHA (5 µg/µl) added at the time of
of Curcuma longa. Turmeric or curcuma, the pow-
seeding (day 0). From day 3 on, 60 U/ml recombinant
dered form of the rhizome, is widely used in Asian IL-2 (rIL-2; Peprotech EC LTD, UK) was added every
countries where this plant has been cultivated for 2–3 days. CD8+ cells from day 0 and day 10–14 were
centuries. Curcumin exhibits a variety of pharma-
isolated by MACS-magnetic cell sorting (Miltenyi Bio-
cological effects including anti-inflammatory, anti-
tec, Medianus, Poland) by positive selection. The pu-
infectious and anticancer activities (Lin et al., 2000). rity was > 97% and cell viability measured by trypan
The exposure of populations worldwide to curcu-
blue exclusion test was over 95%.
cumin-induced death in CD8+ cells
To induce apoptosis with curcumin, CD8+
Western blotting. The protocol used for West-
cells were treated for 8 h with 50 ?M dye (Merck, ern blotting has been reported (Bielak-Mijewska et
Warsaw, Poland). For UVC treatment cells were ir-
radiated with a pulse of ultraviolet light of 254 nm
Proteins (40 ?g per lane) were separated on
and an energy output of 100 J/m2 using a Stratalink-
12% PAGE/SDS and electrotrasferred onto nitrocellu-
er 2400 (Stratagene, La Jolla, CA, USA) and collected lose membrane (Hybond-C, Amersham). Membranes
6 h after irradiation.
were probed overnight at 4oC with rabbit polyclo-
Cell phenotyping. Phenotypes of cells were nal anti-DFF40 (1 : 500), anti-DFF45 (1 : 500), or mouse
examined by standard flow cytometry procedures. monoclonal anti-PARP-1 (1 : 500) (BD Biosciences).
This involved double immunofluorescence staining Specific proteins were visualized with horseradish
of blood samples using Simultest from Beckton-
peroxidase-conjugated anti-IgG antibodies and the
Dickinson. Each experiment included cells incubated enhanced chemiluminescence (ECL) reagent (Amer-
with isotype controls. Samples were analyzed on sham Pharmacia).
FACSCalibur (Becton-Dickinson, Warsaw, Poland) us-
statistics. Statistical analysis was performed
ing Cell-Quest software (Becton-Dickinson).
by using Student’s t-test.
Cell proliferation assay. Mononuclear lym-
phocytes were labelled with a tracker dye, carboxy-
fluorescein diacetate succinimidyl ester (CFSE, Mo-
lecular Probes) according to Hasbold et al. (1999),
before stimulation with PHA and then at the 1st
and 7th day of cell culture as described previously PHA induces cell to proliferation and cell death
(Brzezinska et al., 2003; 2004; Brzezinska, 2005).
DnA content analysis by flow cytometry.
The majority of human lymphocytes isolated
Cells were analyzed for DNA content by flow cy-
from peripheral blood are in a quiescent state. They
tometry. One million cells were collected, washed can be stimulated to proliferate in culture by mi-
and suspended in Nicoletti buffer (0.1% sodium togens and IL-2. Upon stimulation with PHA, which
citrate, pH 7.4, 0.1% Triton X-100, and 50 ?g/ml mimics pathogen activation, T cells undergo intense
propidium iodide). DNA content was determined proliferation and then activation-induced cell death
on a flow cytometer (FACSCalibur, Becton Dick-
(AICD). The most intense proliferation and AICD of
inson). The sub-G1 fraction represents apoptotic T cells were observed at the 7th day following PHA
cells; cellular debris was excluded from the analy-
stimulation (Fig. 1A). After 10 days of culture, pro-
sis. The levels of apoptotic cells induced in specific liferation was still at a high level; almost 70% of live
experimental conditions were calculated according cells were proliferating as measured by CFSE assay.
to the following formula: (percentage of induced Also morphological observation indicated that the
apoptosis minus percentage of spontaneous apop-
majority of cells on day 10–14 of culture were blasts
tosis)/(100 minus percentage of spontaneous apop-
(Fig. 2B). The percentage of dying cells measured
tosis) × 100.
by 7-AAD staining accounted for about 20% at the
Cell viability measurement. Cell viability 5th day after stimulation and lasted at that level to
was measured by MTT (3-(4,5-dimethyldiazol-2-yl)-
the 14th day of culture, but afterwards it increased
2,5-diphenyl tetrazolium bromide; Sigma) assay as dramatically to about 80% in four-week-old cultures
described by the manufacturer.
(Fig. 1B). As the 7-AAD assay does not discrimi-
Spontaneous apoptosis was measured using nate between apoptotic and necrotic cell death, we
7-AAD (7-amino-actinomycin D; Calbiochem) which checked cell DNA content by using propidium io-
stains apoptotic and necrotic cells. The 7-AAD posi-
dide to measure the sub-G1 fraction corresponding
tive cells were analyzed by flow cytometry.
apoptotic cells. The sub-G1 fraction was relatively
Cell morphology observation. Morphologi-
high (about 30%) at 5 days after PHA stimulation
cal observation was performed after Hoechst 33258 and practically absent in two-week-old cultures
staining (Molecular Probes, Eugene, OR, USA). Cells reaching again 20% at the end of culture (4 weeks)
(0.2–0.3 × 106) were centrifuged on cytospin, fixed (Fig. 1B).
with 70% ethanol, washed in PBS and stained for 10
During the initial days of culture the propor-
min in 1 ?M Hoechst 33258 dye. Samples were visu-
tion of cytotoxic and helper cells changed, namely
alized by epifluorescence microscopy (Nicon) and the CD8+ population increased from about 20% on
images were acquired with a color CCD camera.
day 0 to about 60% on day 14 suggesting that CD8+
Caspase-3 activation measurement. The acti-
cells might be less prone to PHA-induced cell death
vation of caspase-3 was analyzed by flow cytometry than CD4+ cells (Fig. 1C).
using PE-conjugated anti-active caspase-3 mAb (BD
Accordingly, for further experiments isolated
Pharmingen) according to the manufacturer’s protocol.
CD8+ cells from day 10–14 of mononuclear lym-
Figure 1. Proliferation, cell death and phenotype of
PHA-stimulated T cells in long term culture of mono-
Figure 2. DnA fragmentation (A) and chromatin con-
densation (B) in quiescent and proliferating CD8+ cells
A. Proliferation assay was performed by cell staining
treated with curcumin or UVC-irradiated.
with CFSE and fluorescence was measured at 5th day af-
DNA fragmentation was assessed by flow cytometry (sub-
ter staining only in the gate of live cells (7-AAD negati-
G1). Data are expressed as mean ± S.D. of three indepen-
ve). B. The amount of dying cells was assessed by 7-AAD
dent experiments. Chromatin condensation was observed
staining and DNA content was measured with propidium
microscopically after staining of cells with Hoechst 33258.
Representative pictures of three independent experiments
1 fraction). C. Cells were phenotyped using
Simultest. Data are expressed as mean ± S.D. of eight in-
To this end, CD8+ cells either isolated directly from
phocyte culture were chosen as they predominate in non-stimulated peripheral lymphocytes (quiescent)
culture during the period of the more intense prolif-
or from cultured mononuclear human lymphocytes
eration and they display the lowest ratio of the cell stimulated with PHA in vitro and cultured for 10–14
death. CD8+ cells activated stimulated after isolation days (proliferating) were irradiated with UVC and
from the mononuclear fraction did not respond with analyzed 6 h later or treated with curcumin for 8 h.
proliferation (not shown).
We chose 50 ?M concentration of curcumin which
had earlier been found to induce in human T cells
UVC but not curcumin induces DNA fragmentation
and Jurkat cells the mode of cell death without oligo-
in CD8+ cells
nucleosomal DNA degradation (Piwocka et al., 1999;
Bielak-Zmijewska et al., 2000; Sikora et al., 2006).
As previously we showed that rat and human
Data presented in Fig. 2A show that 6 h af-
quiescent T cells are less prone to undergo cell death ter UVC irradiation more that 40% of proliferating
induced by UVC than proliferating lymphocytes CD8+ cells revealed significant DNA fragmentation
(Radziszewska et al., 1999; 2000; Piwocka et al., 1999) and were present in the sub-G1 fraction. However,
now we decided to study this difference in more de-
the sub-G1 fraction of curcumin-treated cells did not
tailed by focusing on UVC and curcumin’s impact exceed 5% in either quiescent or proliferating cells.
on non-activated and activated purified CD8+ cells. Similarly only a few percent of quiescent UVC-treat-
cumin-induced death in CD8+ cells
ed cells were found in the sub-G1 subpopulation
Apoptotic chromatin condensation temporally
correlates with DNA fragmentation and frequently
its efficiency depends on the degree of internucleo-
somal cleavage (Widlak et al., 2003). Thus, we have
analyzed nuclear morphology of CD8+ cells treated
with curcumin or UVC-irradiated (Fig. 2B). Only
proliferating UVC-irradiated cells revealed charac-
teristic chromatin condensation and fragmentation
as well as formation of apoptotic bodies resembling Figure 4. Viability of quiescent and proliferating CD8+
typical apoptosis, which could be expected from cells after treatment with 50 µM curcumin.
DNA content analysis. Quiescent CD8+ cells after Cell viability was measured after 8 h of curcumin treat-
UVC irradiation seemed to be quite healthy. Cur-
ment by the MTT assay (values are in the relation to
cumin treatment resulted in chromatin condensation untreated control). Data are expressed as mean ± S.D. of
in almost all cells, quiescent or proliferating. How-
three independent experiments. **, P ? 0.001; ***, P ? 0.0001.
ever, unlike in UVC-irradiated proliferating cells, no flow cytometry (Fig. 3A). As expected the analysis
apoptotic bodies were seen (Fig. 2B).
revealed the presence of active caspase-3 in prolif-
erating, but not quiescent CD8+ cells irradiated with
Curcumin induces caspase-3 activation followed by
UVC. Moreover, active caspase-3 was detected also
cleavage of its substrates
in curcumin-treated cells, both quiescent and prolif-
erating. The activity of caspase-3 was assessed in the
In the majority of apoptotic cells oligonucleo-
same cells by detection of the 89 kDa fragment of
somal DNA degradation results from the activity of PARP-1, which is a specific product of caspase-3 ac-
endonuclease DFF40/CAD. The nuclease is activated tivity (Fig. 3B). The truncated form of PARP-1 could
upon caspase-3-catalyzed cleavage of its inhibitor be detected in proliferating CD8+, both irradiated
DFF45/ICAD (Enari et al., 1998; Halenbeck et al., with UVC and treated with curcumin, as well as in
1998; Liu et al., 1998). Thus, one could conclude from quiescent curcumin-treated cells. Using MTT assay
the results shown above that curcumin, in contrast to we showed that the percentage of survivals account-
UVC, did not induce caspase-3 activation. To verify ed for slightly more than 40% and 80% in curcumin-
this assumption the presence of activated caspase-3 treated proliferating and quiescent cells, respectively
was analyzed in curcumin-treated or UVC-irradiated (Fig. 4). These data show that in terms of survival,
CD8+ either quiescent or proliferating cells using the impact of curcumin is stronger on proliferating
than on quiescent CD8+ cells.
Curcumin induces degradation of DFF45/ICAD that
leads to formation of the potentially active DFF40/
CAD nuclease in CD8+ cells
In healthy non-apoptotic cells, the DFF40/
CAD nuclease exists in the nucleus as a heterodim-
Figure 3. Active caspase-3 in quiescent and proliferating
CD8+ cells at 8 h after treatment with 50 µM curcumin.
(A) The number of cells that contained active form of cas-
pase-3 was measured by flow cytometry and results are
Figure 5. levels of DFF subunits in CD8+ cells after 8 h
expressed as mean ± S.D. of three independent experi-
of treatment with 50 ?M curcumin.
ments. (B) The truncated form of PARP-1 was assessed in
DFF40/CAD (A) and DFF45/ICAD (B) were assessed by
whole cell lysates by Western blotting. A representative
Western blotting in whole cells lysates. The representative
blot of three independent experiments is shown.
blots of three independent experiments are shown.
er with its inhibitor DFF45/ICAD (a 35-kDa splic-
pro-survival and apoptotic pathways is inhibited by
ing variant of DFF45-DFF35/ICAD-S-resides in the curcumin (Deeb et al., 2003; 2004). Recently curcu-
cytoplasm). Activation of caspase-3 results in the min has been shown to repress histone acetyltrans-
cleavage of DFF45/ICAD and release of DFF40/
ferase-dependent chromatin transcription by inhibit-
CAD, which forms active homo-oligomers (Liu et ing its p300/CREB-binding protein (Balasubraman-
al., 1999; Widlak et al., 2003; Woo et al., 2004). Here yam et al., 2004). p300 is a ubiquitously expressed
we show that DFF40/CAD is present in CD8+ cells, global transcriptional coactivator that has a critical
and its total level remains essentially unchanged in role in a wide variety of cellular phenomena includ-
cells treated with UVC or curcumin as measured by ing cell cycle control, differentiation and apoptosis
Western blotting in whole cell lysates (Fig. 5A). In (Giordano & Avantaggiati, 1999).
a marked contrast to the level of DFF40/CAD, the
An open question is the curcumin’s selectivity
level of DFF45/ICAD decreased in curcumin-treated for transformed cells; alas, the data regarding curcu-
CD8+ cells, the decrease being less pronounced in min impact on normal cells are rather scarce. Previ-
quiescent cells than in proliferating ones (Fig. 5B). It ously we showed that curcumin induced cell death
is of note, that in quiescent CD8+ cells, the band of in rat splenocytes as well as in human quiescent
DFF35 was not present, but in proliferating cells, as and proliferating T cells, but the cell death mode
expected, its disappearance was proportional to the in terms of chromatin degradation and oligonucleo-
decrease of the DFF45 band.
somal DNA fragmentation did not resemble typical
apoptosis (Bielak-Zmijewska et al., 2000). Also oth-
ers described cell death in human V?9V?2 T cells
caused by curcumin, due to high molecular mass,
but not oligonucleosomal DNA degradation (Cipri-
There are many facets to cancer prevention ani et al., 2001). Recently, the Gautam’s group (Gao
and one of them is using natural or synthetic com-
et al., 2004) published data of an immunomodula-
pounds allowing suppression, retardation or inver-
tory activity of curcumin, namely its suppression of
sion of carcinogenesis. Only a few such agents have lymphocyte proliferation, development of cell-medi-
been used to date in the clinic and these include ated cytotoxicity and cytokine production in vitro.
non-steroidal anti-inflammatory drugs for colon, fi-
Although curcumin at 30 ?M concentration irrevers-
nasteride for prostate, and tamoxifen or reloxifene ibly inhibited proliferation of splenocytes it did not
for breast tumors. An ideal chemopreventive agent affect cell viability measured by MTT test (Gao et
should restore normal growth control to preneoplas-
al., 2004). In our hands 50 ?M curcumin decreased
tic or cancerous cells by modifying aberrant signal-
proliferating cell viability to 40% already at 8 h as
ling pathways or inducing cell death in cells beyond measured by MTT test. Also in a lower concentra-
repair. The characteristics of such an agent include tion, namely 25 ?M, curcumin was harmful to nor-
selectivity for transformed cells and more than one mal T cells (not shown).
mechanism of action to foil the redundancy or cross-
It is believed that the side effects of anticancer
talk in signalling pathways (Manson et al., 2005). It therapy affect mainly proliferating cells. Indeed, pre-
seems that curcumin fits this picture very well by viously (Radziszewska et al., 1999) and in this paper
affecting many signalling pathways, it is able to in-
we showed that UVC, which is a known DNA dam-
duce cell death in any tested cancer cells like leukae-
aging agent, did not affect quiescent T cells, but in-
mia, melanoma, breast, lung, prostate, colon, renal, duced apoptosis in proliferating ones. In contrast to
hepatocellular and ovarian carcinomas (reviewed in UVC, curcumin induces cell death not only in prolif-
Karunagaran et al., 2005), including those resistant to erating but also in quiescent CD8+ cells, however, in
apoptosis due to the multidrug resistance phenotype a short-time treatment proliferating cells seem more
(MDR) (Bielak-Mijewska et al., 2004).
sensitive to curcumin than quiescent ones. Moreover,
It appears from the available data that curcu-
the cell death mode induced by curcumin is distinct
min impact on cancer cells can be explained by its from that attributable to classical apoptosis whose
pleiotropic activity and many targets in the cell like main hallmark is oligonucleosomal DNA fragmenta-
COX-2 (Goel et al., 2001; Shishodia et al., 2003), HO-1 tion resulting from caspase-3 activation.
(Balogun et al., 2003) or v-Src (Leu et al., 2003). On
Activation of caspase-3 led to proteolysis of
the other hand, the multitiude of curcumin’s targets its specific substrates including DFF45/ICAD, the in-
can also be explained by its influence on transcrip-
hibitor of the major apoptotic nuclease DFF40/CAD.
tion regulation. Curcumin has been shown to inhibit One would assume that the release of the nuclease
the AP-1 transcription factor which is involved in from its inhibitor should result in DNA cleavage.
apoptotic program and regulation of cell prolifera-
However, DNA fragmentation was not observed in
tion of many cells (Sikora et al., 1997; Bharti et al., curcumin-treated cells. Essentially the same effect
2004; Zheng et al., 2004). Also NF-?B involved in was observed earlier in Jurkat cells. We have some
cumin-induced death in CD8+ cells
evidence that curcumin can inhibit DFF40/CAD by were detected in blood or urine (Sharma et al., 2001).
blocking magnesium binding in the active centre, Nonetheless, considering curcumin or curcumin de-
thus preventing DNA fragmentation but not affect-
rivatives’ future use in the clinic and eventual in-
ing cell death itself (Sikora et al., 2006).
travenous application adverse side-effects could be
Curcumin is a very promising chemopreven-
expected such as de