Induction of apoptosis in human lung cancer cells by curcumin

Cancer Letters 208 (2004) 163–170
www.elsevier.com/locate/canlet
Induction of apoptosis in human lung cancer cells by curcumin
G. Radhakrishna Pillai1, Anand S. Srivastava1, Tarek I. Hassanein,
Dharam P. Chauhan, Ewa Carrier*
Department of Medicine, Pediatrics and Family and Preventive Medicine, School of Medicine, University of California,
San Diego, 9500 Gilman Drive, La Jolla, CA 92093-0062, USA
Received 14 July 2003; received in revised form 24 September 2003; accepted 12 January 2004
Abstract
Curcumin, a phenolic compound from the rhizome of the plant Curcuma longa has anti-in?ammatory, antioxidant and anti-
cancer activities. Although the precise mode of action of this compound is not yet elucidated, studies have shown that chemo-
preventive action of curcumin might be due to its ability to induce apoptosis and to arrest cell cycle. This study investigated
the cellular and molecular changes induced by curcumin leading to the induction of apoptosis in human lung cancer cell
lines—A549 and H1299. A549 is p53 pro?cient and H1299 is p53 null mutant. The lung cancer cells were treated with
curcumin (0 – 160 mM) for 12 – 72 h. Curcumin inhibited the growth of both the cell lines in a concentration dependent manner.
Growth inhibition of H1299 cell lines was both time and concentration dependent. Curcumin induced apoptosis in both the lung
cancer cell lines. A decrease in expression of p53, bcl-2, and bcl-XL was observed after 12 h exposure of 40 mM curcumin. Bak
and Caspase genes remained unchanged up to 60 mM curcumin but showed decrease in expression levels at 80 – 160 mM. The
data also suggest a p53 independent induction of apoptosis in lung cancer cells.
q 2004 Published by Elsevier Ireland Ltd.
Keywords: Curcumin; Lung cancer; bcl-2; bclXL; bak; bax; p53; c-myc; Caspase; PARP
1. Introduction
widely used as a coloring and ?avoring agent in food
[1]. Its anti-in?ammatory activity is well documented.
Cancer causes signi?cant morbidity and mortality
Curcumin is not toxic to mammals at very high doses
(5 – 10% by weight of diet) [2].
and is a major public health problem worldwide. An
Curcumin is found to have inhibitory function
effective cancer prevention program, diet, and exer-
towards a broad range of tumors such as mammary
cise may decrease the incidence of cancer. Plant-
adenocarcinoma, fore stomach, duodenal and colon
derived compounds are known to have curative
cancer as well as 12-O-tetradecanoyl-13-phorbol ester
potential. Curcumin (diferuloylmethane) is a phenolic
(TPA) induced skin tumors in mice [2,3]. Curcumin is
compound from the plant Curcuma longa (Linn). It is
a potent anti-cancer agent and affects cells in a cell
type dependent manner. Its ability to induce apoptosis
* Corresponding author. Tel.: þ1-858-822-1050; fax: þ1-858-
in different cancer cells indicates the possibility of
534-7340.
developing curcumin as a universal cancer prophy-
E-mail address: ecarrier@ucsd.edu (E. Carrier).
1 Both authors contributed equally to this work.
lactic agent.
0304-3835/$ - see front matter q 2004 Published by Elsevier Ireland Ltd.
doi:10.1016/j.canlet.2004.01.008

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G.R. Pillai et al. / Cancer Letters 208 (2004) 163–170
The protein p53 has an important role in patho-
USA) were used for this study. Cell line A549 is p53
genesis of neoplasia [4]. The mechanism involved
wild type and H1299 is devoid of endogenous p53.
entails a rapid increase in p53 protein levels and the
The cells were grown in RPMI1640 culture medium
mediation of several cellular responses including G1
supplemented with 2 mM L-glutamine and 10% FCS,
arrest, DNA damage repair and induction of apoptosis
penicillin (50 IU/ml) and streptomycin (50 (g/ml) at a
[5]. One of the major signaling pathways involved
temperature of 37 8C in a humidi?ed incubator with a
in apoptotic cell death includes the intracellular
5% CO2 atmosphere.
caspases, a family of structurally related cysteine
proteases [6]. Caspase activity is responsible, either
2.2. Curcumin treatment of cells
directly or indirectly, for the cleavage of cellular
proteins, which are characteristically proteolyzed
Curcumin (Sigma Chemical Co, St Louis MO,
during apoptosis. For example, caspases -2, -3, -6,
USA) was dissolved in DMSO at a concentration of
-7 and -9 can cleave poly (ADP ribose) polymerase
10 mM and was stored in a dark-colored bottle at 4 8C
(PARP) [7]. Bcl-2 family proteins are one of the
as a stock solution. The stock was diluted to the
already identi?ed regulators of apoptosis. Bcl-2
required concentration immediately before use with
family of homologous proteins represents a critical
growth media. The cells were exposed to curcumin at
checkpoint within most apoptotic pathways, acting
different concentration (0 – 160 (M) and for different
upstream of such irreversible damage to cellular
time durations (6 – 72 h). Cells grown in media
constituents [8]. At least 15 bcl-2 family members
containing equivalent amount of DMSO without
have been identi?ed so far in mammalian cells. They
curcumin served as control.
function either as proapoptotic (Bax, Bak, Bad) or anti
apoptotic (Bcl-2, Bcl-XL) regulators. These proteins
2.3. Cell viability assay
form heterodimers of anti and pro-apoptotic members,
thereby titrating one another’s function [8]. The ratio
The viability of the cells was assessed by MTT
of anti-apoptotic and proapoptotic proteins deter-
(3,4,5-dimethylthiazol-2-yl)-2-5-diphenyltetrazolium
mines in part how cells respond to apoptotic or
bromide) assay, which is based on the reduction of
survival signals [9]. Despite extensive analysis of
MTT by the mitochondrial dehydrogenase of intact
anti-tumor activities of curcumin, its ability to
cells to a purple formazan product [9,10].
modulate lung cancer growth has not yet been well
Cells (1 £ 105) were plated in a 96-well plate
characterized. We used lung cancer cell lines to study
(Coastar from Corning, NY). After 24 h they were
the effect of different concentrations of curcumin on
treated with different concentrations (0 – 160 (M) of
cell viability and genes related to apoptosis. Our
curcumin for different time intervals (12 – 72 h). After
results demonstrated that curcumin causes growth
the treatment, media containing curcumin were care-
arrest and apoptosis in lung cancer cell lines and the
fully removed by aspiration. Hundred microlitre
growth inhibitory effects of curcumin appeared to be
of 0.5 mg/ml MTT in cell culture medium was
mediated by the regulation of bcl-2, bcl-xL, bax,
added to each well and incubated for 4 h as described
caspase-1, caspase-3, c-myc and p53 genes. Curcumin
in Ref. [10]. Hundred microlitre of 1% SDS was
caused induction of apoptosis and the induction was
added to each well after 4 h. The plates were covered
independent of p53 status of the cell lines.
with aluminum foil and kept in an incubator for
12 h for dissolution of the formed formazan crystals.
Amount of formazan was determined measuring
2. Materials and methods
the absorbance at 540 nm using an ELISA plate
reader.
2.1. Cell lines
2.4. PARP cleavage
The human lung adenocarcinoma cell line A549
(ATCC, Manassas, VA, USA) and the large cell lung
The apoptosis was determined by measuring the
carcinoma cell line H1299 (ATCC, Manassas, VA,
cleavage of poly (ADP) ribose polymerase (PARP).

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G.R. Pillai et al. / Cancer Letters 208 (2004) 163–170
165
PARP is a highly conserved nuclear enzyme present
caspase-3 and c-myc the following primer sequences
in higher eukaryotes. The enzyme is a DNA binding
were used (Table 1). For p53 apoptosis primer kit
protein that recognizes DNA strand breaks and is
(Apoptosis set 1, Biosource Camarillo, CA, USA)
implicated in DNA repair and in the apoptotic
was used. In this experiment, the PCR conditions
response of cells [11]. PARP functions by adding
included an initial denaturation at 96 8C for 1 min
poly (ADP-ribose), in an ATP and NAD þ dependent
followed by two cycles of 96 8C, each for 1 min and
manner, to various proteins in response to DNA
annealing step at 58 8C for 4 min. PCR was carried
damage and has been shown to directly interact with
out for 28 cycles as follows: denaturation at 94 8C
DNA polymerase. As a marker for apoptosis, PARP
for 1 min, annealing and extension step at 58 8C
cleavage has been shown to occur early in the
for 3 min. The ?nal step included incubation at
apoptotic response as a result of caspase-3 activity
70 8C for 10 min followed by a soaking at 25 8C.
[12]. PARP cleavage correlates well with chromatin
Statistical analyses were carried out using ANOVA
condensation and has been shown to be associated
and student’s ‘t’ test.
with the condensed chromatin in apoptotic cells, as a
measure of apoptosis appearing as early as 3 h post
apoptosis inducing event, and precedes the ability to
3. Results
detect actual DNA fragmentation. PARP cleavage
was tested by using an anti PARP (rabbit polyclonal)
3.1. Effect of curcumin on the growth of A549
cleavage site (214/215) speci?c antibody-FITC con-
and H1299 cells
jugate (Biosource, Camarillo, CA, USA). The proto-
col supplied by the manufacturer was strictly
A549 and H1299 lung cancer cell lines were used
followed. After antibody labeling, the cells were
as a model system to examine the effect of curcumin
analyzed by ?ow cytometry (Coulter-Epics XL,
on their growth. The growth inhibitory effect of
Coulter, Krefeld, Germany).
curcumin was concentration dependent in both the
cell lines (Figs. 1 and 2). H1299 cells also showed
2.5. Gene expression studies
time dependent effect of curcumin and were more
sensitive to curcumin treatment than A549 cells. The
Expression of apoptosis related genes, bcl2, bcl-XL,
IC50 s at 24 exposure of curcumin were 50 and
bax, bak, caspase-3, p53 and c-myc was studied using
40 mM for A549 and H1299 cells, respectively.
reverse transcriptase-PCR (RT-PCR). The housekeep-
ing genes bactin/GAPDH were used as control. Total
3.2. Induction of apoptosis
RNA was isolated using trizol reagent (Invitrogen,
Carlsbad, CA, USA). cDNA was generated by reverse
We analyzed PARP cleavage in H1299 cells by
transcription of 3 mg of total denatured RNA. Reverse
?ow cytometry. PARP cleavage site-speci?c MoAab
Transcription was performed at 37 8C for 1 h using
(rabbit polyclonal anti PARP cleavage site (214/215)
MMLV Reverse Transcriptase kit (GIBCO/BRL) in a
speci?c antibody-FITC conjugate (Biosource Inter-
total mixture of 15 ml. Two microlitres from the above
national, USA)) was used. The data (Fig. 3) correlated
RT product was used for PCR reaction. PCR was
with the results of cell viability assays (Figs. 1 and 2).
carried out using the gene speci?c upstream and
PARP cleavage started at a lower concentration
downstream primers. Initial denaturation at 95 8C for
(10 mM) and increased with the increasing concen-
3 min was followed by a PCR cycle of denaturation
tration of curcumin. At 24 h, a concentration of
at 95 8C for 45 s, annealing at 69 8C for 1 min and
100 mM of curcumin induced PARP cleavage in
strand extension at 72 8C for 1 min. Number of cycles
, 90% of treated cells (Fig. 3).
were 30 for bcl2, c-myc, and caspase 1 and 26 for
bactin and bax. PCR products were separated on a
3.3. Expression of apoptotic genes
1.8% agarose gel and stained with ethidium bromide.
The relative expression of mRNA was quantitated by
Effect of curcumin on the mRNA expression of
densitometry [13]. For b-actin, bcl-2, bcl-xL, bax, bak,
selected apoptosis related genes was analyzed by

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Table 1
4. Discussion
PCR primers used in the gene expression studies
b-actin
(accession number X00351)
Our results, for the ?rst time, demonstrate that
Forward (nt 936-955)
curcumin induces apoptosis in human lung cancer
50-CTGTCTGGCGGCACCACCAT-30
cells. Curcumin is a hydrophobic molecule and passes
Reverse (nt 1170-1189
easily through the plasma membrane into the cytosol
50-GCAACTAAGTCATAGTCCGC-30
[14]. The results of this study demonstrate that this
phenolic substance has the ability to reduce the
Bcl-2
(accession number M13994)
Forward (nt1799-1823) 50-
viability of lung cancer cells through induction of
AGATGTCCAGCCAGCTGCACCTGAC-30
apoptosis. Forty to ?fty micromoles of curcumin
Reverse (nt 2139-2165) 50-
decreased the cell viability to 50%, whereas 160 mM
AGATAGGCACCCAGGGTGATGCAAGCT-30
Curcumin reduced the viability by 95%. It is known
that curcumin is poorly absorbed and it will not be
Bax
(accession number L22473)
Forward (nt 172-195)
possible to achieve these high levels of curcumin in
50-AAGCTGAGCGAGTGTCTCAAGCGC-30
blood [15]. Therefore, the studies should also be
Reverse (nt 516-537)
concentrated on increasing the bioavailability of
50-TCCCGCCACAAAGATGGTCACG-30
curcumin [15]. This induction of apoptosis occurred
within several hours; consistent with the view that
Bak
(accession number X84213)
curcumin induces apoptosis by activating the pre-
Forward (nt 23-47)
50-TCCAGATGCCGGGAATGCACTGACG-30
existing apoptosis machinery. The cell line, H1299, is
Reverse (nt 1191-1214) 50-
p53 de?cient and A 549 is p53 pro?cient. Inhibition of
TGGTGGGAATGGGCTCTCACAAGG-30
growth of both cell lines (p53 de?cient and pro?cient)
suggest that growth inhibitory effect of curcumin is
Bcl-xL
(accession number Z23115)
independent of p53 expression. Induction of apoptosis
Forward (nt 381-402)
50-ATGGCAGCAGTAAAGCAAGCGC-30
in both the cell lines also suggest that a p53
Reverse (nt 903-922)
independent pathway is operative in this system.
50 TTCTCCTGGTGGCAATGGCG-30
Caspase-3
(CPP32, yama, apopain) (accession number U26943)
Forward (nt 340-361)
50-TTTGTTTGTGTGCTTCTGAGCC-30
Reverse (nt 720-739)
50-ATTCTGTTGCCACCTTTCGG-30
C-myc
(accession number V00568)
Forward (nt 948-967)
50-CCAGGACTGTATGTGGAGCG-30
Reverse (nt 1433-1452)
50-CTTGAGGACCAGTGGGCTGT-30
RT-PCR. A decrease in expression of p53, bcl-2, and
bcl-XL was observed. A 12 h exposure to 40 mM
of curcumin decreased the expression of BclXL by
40%, Bcl-2 by 35% and p53 by 25% of normal levels
Fig. 1. Effect of curcumin on cell viability. A549 cells were plated
(Fig. 4). Exposure to 160 mM of curcumin for 12 h
on 96 well plates and on the second day, cells were treated with
decreased expression of these genes to below
different concentration of curcumin for different time periods.
Experiments were done in triplicate. The cell viability was then
detectable levels. Bak, and Caspase genes remained
determined by MTT assay, as described in Section 2. Values
unchanged up to 60 mM curcumin but showed
represent mean (^ SD) cell viability as a percentage of untreated
decrease in expression levels at 80 – 160 mM.
control samples.

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167
p53 and a presence of mutant p53 renders various cell
lines resistant to the chemotherapeutic agents or
ionizing radiation [23].
In our experiments, curcumin showed increased
expression of c-myc, another immediate early gene,
intimately involved in the control of cell growth and
apoptosis [24]. The proto-oncogene c-myc is known
to regulate neoplastic development and apoptotic cell
death, and a relationship between p53 abnormalities
and c-myc activation has been suggested previously
[25]. There is a controversy regarding the relationship
between p53 and c-myc in the process of apoptosis.
For example, several investigators have reported that
c-myc-mediated apoptosis requires wild-type p53
[26]. In contrast, other investigators reported that
c-myc-mediated apoptosis does not requires wild type
Fig. 2. Effect of curcumin on cell viability. H1299 cells were plated
p53 [27]. The exact mechanism of c-myc-mediated
on 96 well plates and on the second day, cells were treated with
apoptosis via the p53 independent pathway is
different concentration of curcumin for different time durations.
unknown.
Experiments were done in triplicate. The cell viability was then
In order to clarify whether cell death following
determined by MTT assay, as described under Section 2. Values
curcumin treatment is by necrosis or apoptosis, we
represent mean (^ SD) cell viability as a percentage of untreated
control samples.
studied the PARP cleavage. Caspases are activated
during apoptosis and that, in-turn cleave PARP. PARP
It has been reported that when p53 gene is mutated in
cleavage is known to be a good index for apoptosis
A549 cells, it has lost the pro-apoptotic function [16].
[12]. The stability of p53 is regulated by several
Strasser et al. reported the existence of a p53
factors. The interaction of p53 with PARP represents
independent pathway in leukemic and lymphatic cell
one of several alternative pathways regulating
lines [17]. In a previous report a growth arrest and
apoptosis of B cell lymphoma by down regulation of
c-myc, bcl-XL and p53 on exposure to curcumin was
documented [18]. Hipp and Baver [19] reported that
p53 is not necessary for intercellular induction of
apoptosis, as p53 negative transformed cells are
sensitive to intercellular induction of apoptosis and
p53 negative non-transformed cells are able to induce
apoptosis. It was also reported in human breast cancer
cell line that CD437 induces G0-G1, arrest and
apoptosis via regulation of p21WAFI/CIPI, Bcl-2 and
Bax in a p53 independent manner [20]. Adachi et al.
and Miyashita et al. reported that CD437-induced
apoptosis in some lung cancer cells did not necessarily
require p53 [20 – 22]. It therefore appears that in
Fig. 3. Determination of PARP cleavage in curcumin treated H1299
certain cells apoptosis can be induced in a p53
lung cancer cells by ?ow cytometry. 1 £ 106 H1299 cells were
independent pathway either by inducing downstream
plated in a T25 ?ask and on the second day they were treated with
genes of p53 (e.g. p21, Bax etc.) or by an as yet
different concentrations (0, 1, 5, 10, 20, 40, 60, 80 and 100 mM) of
curcumin for 24 h. After 24 h cells (adherent as well as ?oating)
unknown mechanism. It was also reported that mutant
were harvested by centrifugation and analyzed by ?ow cytometry to
p53 has a dominant negative effect on a wild type
detect the extent of PARP cleavage, as described in Section 2.

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G.R. Pillai et al. / Cancer Letters 208 (2004) 163–170
the caspases activated DNase (CAD/CPAN). Once
the ICAD is cleaved, CAD enters the nucleus and
degrades chromatin into oligonucleosomal fragments
[32]. This, in turn leads to apoptosis.
We observed a down-regulation of bclXL gene. It is
reported that bclXL is down-regulated by c-myc
protein [33,34]. Since we observed an increase in
c-myc expression, the role of c-myc in the down
regulation of bcl XL can be postulated. Bcl XL is an
anti-apoptotic gene and hence its down-regulation is
associated with apoptosis induced by curcumin
[35,36]. The effect of curcumin on the bcl-2 gene
appears to be less pronounced. Proteins of the bcl-2
family turned out to be important regulators of
apoptosis [37]. This model demonstrated that homo-
Fig. 4. Expression of pro- and anti-apoptotic genes in A549 cells
dimers of death agonists induced apoptosis, while
following exposure to curcumin for 12 h. Cells were plated on T25
hetero-dimers or homo-dimers of death antagonists
plates and after 24 h they were treated with different concentrations
prevented apoptosis [38]. Several studies indicated
of curcumin. After curcumin treatment cells were harvested and
that these pro- and anti-apoptotic proteins (Bcl-2, bcl
RNA was isolated using trizol. RT-PCR was performed as described
XL and bax) might function independently and
in Methods. PCR products were resolved on a 1.8% agarose gel and
visualized using ethidium bromide. Column 1, control cells treated
without formation of hetero-dimers [39]. The high
with vehicle; Column 2, 10 mM of curcumin treated cells; Column
basal level of bcl-2 expression is consistent with p53
3, 20 mM; Column 4, 40 mM; Column 5, 60 mM; Column 6, 80 mM
inactivation because p53 inhibits bcl-2 gene
and Column 7, 160 mM curcumin.
expression [22]. A high level of apoptosis, even in
the presence of higher basal level of bcl-2 may
the stability of p53 protein. PARP forms tight
suggest the possibility of a treatment-associated
complexes with p53 and colocalizes with wild-type
phosphorylation of that protein. Treatment associated
as well as with mutant p53 protein [28,29]. The result
bcl-2 phosphorylation, was reported to cause a loss of
of this study con?rmed curcumin-induced apoptosis
its anti-apoptotic function [40].
in lung cancer cell lines.
In summary, we have demonstrated that the cancer
To obtain further insight into the mechanism of
chemopreventive agent curcumin induces apoptosis in
curcumin action, we studied the expression of caspse-
lung cancer cell lines. Induction of apoptosis by
3, a major player in apoptotic cell death. Results of
curcumin involves multiple pathways. Curcumin up-
this experiment suggest the possibility that the
regulates different pro-apoptotic genes and at the
mechanism of curcumin-induced apoptosis in cancer
same time down-regulate some of the anti-apoptotic
cells involve caspase-3 activation and the resultant
genes. Homo-dimerisation or hetero-dimerisation of
cascade of reactions. Caspases have been shown to be
this pro- and anti-apoptotic genes in favor of apoptosis
activated during apoptosis in many cell systems and
is one of the suggested mechanisms of curcumin-
induced apoptosis.
play critical roles in both the initiation and the
execution of apoptosis [30]. Recently it was reported
that caspase-3 is essential for DNA fragmentation and
the morphological changes associated with apoptosis
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Document Outline

• Induction of apoptosis in human lung cancer cells by curcumin
  • Introduction
  • Materials and methods
    • Cell lines
    • Curcumin treatment of cells
    • Cell viability assay
    • PARP cleavage
    • Gene expression studies
  • Results
    • Effect of curcumin on the growth of A549 and H1299 cells
    • Induction of apoptosis
    • Expression of apoptotic genes
  • Discussion
  • References

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