Nihms 16826

NIH Public Access
Author Manuscript
Cancer Biol Ther. Author manuscript; available in PMC 2007 April 17.
Published in final edited form as:
NIH-PA Author Manuscript
Cancer Biol Ther. 2007 February ; 6(2): 178–184.
Curcumin Induces G2/M Arrest and Apoptosis in Cisplatin-

Resistant Human Ovarian Cancer Cells by Modulating Akt and p38
MAPK
Nathan M. Weir1,¥, Karuppaiyah Selvendiran1,¥, Vijay Kumar Kutala1, Liyue Tong1, Shilpa
Vishwanath1, Murugesan Rajaram1, Susheela Tridandapani1, Shrikant Anant2, and
Periannan Kuppusamy1,*
1 Davis Heart and Lung Research Institute and Comprehensive Cancer Center; Department of Internal
Medicine; Ohio State University; Columbus, Ohio USA
2 University of Oklahoma; Norman, Oklahoma USA
Abstract
Curcumin, a major active component of turmeric, is known to induce apoptosis in several types of
cancer cells, but little is known about its activity in chemoresistant cells. Hence, the aim of the present
study was to investigate the anticancer properties of curcumin in cisplatin-resistant human ovarian
cancer cells in vitro. The results indicated that curcumin inhibited the proliferation of both cisplatin-
resistant (CR) and sensitive (CS) human ovarian cancer cells almost equally. Enhanced superoxide
generation was observed in both CR and CS cells treated with curcumin. Curcumin induced G2/M
phase cell-cycle arrest in CR cells by enhancing the p53 phosphorylation and apoptosis through the
activation of caspase-3 followed by PARP degradation. Curcumin also inhibited the phosphorylation
of Akt while the phosphorylation of p38 MAPK was enhanced. In summary, our results showed that
curcumin inhibits the proliferation of cisplatin-resistant ovarian cancer cells through the induction
of superoxide generation, G2/M arrest, and apoptosis.
Keywords
ovarian cancer; curcumin; superoxide; cell cycle; apoptosis; Akt
INTRODUCTION
Ovarian cancer is the most commonly diagnosed and lethal gynecological malignancy in the
United States and Europe.1,2 The high mortality rate is attributed to the lack of early detection
and also due to the development of chemoresistance.3,4 Although platinum-based compounds
such as cisplatin, in combination with taxanes are initially effective, the five-year survival rates
are only about 50%.5 Despite the fact that most of the ovarian tumors are sensitive to
chemotherapy for the first time, the development of recurrent tumors that are resistant to
cisplatin remains a major hurdle to successful therapy and is responsible for poor long-term
overall survival.5,6 Cisplatin resistance is associated with defects in the apoptotic pathway
including p53 and Bcl-2 family members and death receptors.7 Cisplatin resistance is also
associated with the altered activation of signaling pathways which include PI3K/Akt,8
MAPK9 or JAK/STAT.10 Another suggested mechanism for the drug resistance is the increase
*Correspondence to: Periannan Kuppusamy; Ohio State University; 420 West 12th Ave, Room 114; Columbus, Ohio 43210 USA; Tel.:
614.292.8998; Fax: 614.292.8454; Email: kuppusamy.1@osu.edu.
†These authors contributed equally to this paper.
Weir et al. Page 2
in intracellular thiols in the redox pathway, which may inactivate and remove the platinum
compounds.11,12 Several groups have targeted this redox pathway in an attempt to circumvent
the thiol-induced resistance.13 Recently, we observed that a nitroaspirin derivative
(NCX-4016, an NO donor) partially restored the cisplatin sensitivity by depleting thiols.14
Curcumin (diferuloylmethane) is a major constituent of turmeric powder which is extracted

from the rhizomes of the plant Curcuma longa found in south and southeast tropical Asia. It
has been widely used in Asian countries for centuries in daily cooking preparations.15 The
medicinal value of curcumin has been well recognized for its anti-inflammatory, antimicrobial,
wound healing, and anti-tumor activities.16–19 Several studies have indicated that people in
southeastern Asian countries have a much lower risk of acquiring colon, gastrointestinal,
prostate, breast, ovarian, and other cancers than Western populations.20,21 It is likely that
constituents of their diets such as curcumin, garlic, ginger, chillies, etc., may play a role in the
prevention of such cancers.21 Studies have shown the chemopreventive properties of curcumin
from human malignances.22–24 Clinical evaluations of curcumin as a chemopreventive agent
for many cancers including breast, prostate, colon, and lung have been carried out.25,26 The
anti-carcinogenic properties of curcumin in animal models have been demonstrated.27,28
However, the molecular mechanism underlying curcumin’s chemopreventive effect has not
been fully elucidated, although several mechanisms have been proposed.29
Cell cycle inhibition and the induction of apoptosis are common mechanism(s) proposed for
the anticancer effects of curcumin.18,24,30,31 Recent studies have shown that curcumin is a
potent inhibitor of tumor initiation in vivo.32,33 The antiproliferative and apoptotic effects of
curcumin on tumor cells in vitro have also been reported.34 Curcumin has been shown to induce
apoptosis of cancer cells via inhibition of NFκB and STAT3.32,33 Curcumin has also been
shown to suppress the expression of various NFκB-regulated genes, including Bcl-2, COX-2,
cyclin D1 and adhesion molecules.35 The anticancer activity of curcumin has recently been
attributed to the modification of thioredoxin reductase, an enzyme that plays a key role in
modulating the redox pathway.36 Although the anti-cancer effects of curcumin have been
studied in various cancer cells, it remains unknown whether curcumin possesses anticancer
effects on drug-resistant cells such as cisplatin-resistant ovarian cancer cells. Therefore, the
aim of this study was to determine whether curcumin shows cytotoxic effects in cisplatin-
resistant human ovarian cancer cells and to elucidate the mechanism of its action. Our results
showed that (1) curcumin enhanced superoxide generation in ovarian cancer cells; (2) curcumin
induced cell cycle arrest and apoptosis in cisplatin-resistant ovarian cancer cells; (3) the
curcumin-induced apoptosis in cisplatin-resistant cells was mediated through the inhibition of
Akt and an increase in the activation of p38 MAPK and p53.
MATERIALS AND METHODS

Reagents and culture materials

Curcumin ((1,7-bis[4-hy-droxy-3-methoxyphenyl]-1,6-heptadiene-3,5-dione), GSH, (L-
glutamyl-L-cysteinylglycine), DMSO (dimethyl sulfoxide) and MTT [3-(4,5-
dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] were obtained from Sigma (St Louis,
MO). Cell culture medium (RPMI 1640), fetal bovine serum, antibiotics, sodium pyruvate,
trypsin, and phosphate-buffered saline (PBS) were purchased from Gibco, BRL (Grand Island,
NY). Polyvinylidene fluoride membrane (PVDF) (Millipore), and molecular weight marker
were obtained from Bio-Rad, and Fluoromount-G from Southern Biotech. Lab-Tek II chamber
slides were purchased from Nalge Nunc International (Naperville, IL). Antibodies against poly-
adenosine diphosphate ribose polymerase (PARP), Bax, p38 MAPK, caspase-7 and cleaved
caspase-3 (19 kDa and 17 kDa) and cleaved caspase-7 (20 kDa) were purchased from Cell
Signaling Technology (Beverly, MA), and Akt (Ser473) and p53 from Santa Cruz
Biotechnology (Santa Cruz, CA). RNase was from Promega Corporation (Madison, WI).
Weir et al. Page 3
Enhanced chemiluminescence (ECL) reagents were obtained from Amersham Pharmacia

Biotech (Buckinghamshire, UK). All other reagents and compounds were analytical grades.
Cells and culture conditions

Cisplatin-resistant (CR) and cisplatin-sensitive (CS) human ovarian cancer cells were obtained
from Dr. Sridhar (Howard University Medical School, Washington DC). Cells were grown in
RPMI 1640 medium supplemented with 10% FBS, 2% sodium pyruvate, 1% penicillin and
1% streptomycin. Cells were grown in T-75 flasks to 80% confluence at 37°C in an atmosphere
of 5% CO2 and humidified air. Cells were routinely trypsinized (0.05% trypsin/EDTA) and
the cell count was determined by using a NucleoCounter, automated cell counter, (New
Brunswick Scientific, Edison, NJ).
Cell proliferation assay

Cell proliferation was determined using the conversion of MTT to formazan via mitochondrial
oxidation. Cells were grown in T-75 flasks to >80% confluence. They were then trypsinized,
counted, and seeded in 96-well plates with an average of 7,000 cells/well. Cells were incubated
overnight and then treated in triplicate with 10 or 50 uM curcumin for 12 or 24 h. All
experiments were repeated at least three times.
Superoxide determination by DHE fluorescence

Cells were seeded in Chamber slides with 1 x 105 cells per chamber and incubated overnight.
The following day, cells were preincubated with dihydroethidium (DHE, 10 μM) for 1 h. The
cells were then washed with PBS and treated with varying doses of curcumin for 4 h. After
incubation, the cells were washed and fixed with 3% paraformaldehyde supplemented with
McIIvaine’s buffer for 15 min at 4°C. The paraformaldehyde was then washed off and a cover
slip was fixed using Fluoromount-G. Red fluorescence, indicating superoxide formation, was
detected on a Nikon Eclipse TE2000-U, using excitation/emission at 488/585 nm. Images (12
bit) were acquired using a 400 ms exposure time and the fluorescence intensity was quantified
by MetaMorph software. The intensity was calculated as an average of four areas with a similar
number of cells.
GSH assay
Intracellular levels of GSH were determined by spectrophotometry using DTNB (5,5'-dithiobis
(2-nitrobenzoic acid) (Ellman’s reagent, Cayman Chemical Co., Ann Arbor, Michigan) which
produces a yellow color with GSH to yield TNBA (5-thio-2- nitrobenzoic acid). Cell extracts
were treated with phosphoric acid, the precipitated proteins were centrifuged, and the
supernatants were treated with triethanolamine to bring to neutral pH and then the DTNB
reagent was added and the resulting solution was measured using a 96-well plate ELISA reader
(Beckmann Coulter, AD 340) at 405 nm. All experiments were run in at least four parallels
and repeated thrice. The concentration of GSH was determined from a standard curve prepared
with known concentrations of GSH under similar conditions.
Cell cycle analysis by flow cytometry

Curcumin-treated cells were harvested, fixed overnight with 75% ethanol at −20°C, washed
three times with PBS, treated with RNase A (1 μg/ml), and then stained with propidium iodide.
After propidium iodide staining, the cells were analyzed by flow cytometry (Becton Dickinson,
Franklin Lakes, NJ). The percentage of cells in the G2/M and sub-G1 apoptotic cell population
was determined using CELLQuest software (Becton Dickinson).
Weir et al. Page 4
Immunoblot analysis
Cells were lysed in RIPA buffer, phenyl-methylsulphonyl fluoride (PMSF, 0.1 mM), sodium
orthovanadate (1 mM), and aprotinin and leupeptin (2 μg/ml). The lysate was centrifuged at
12000 x g for 20 min at 4°C and the supernatant was removed. The protein concentration was
measured using a Bio-Rad protein assay kit. After boiling for 10 min in the presence of 2-
mercaptoethanol, samples containing cell lysate protein were separated on a 10 or 15% sodium
dodecyl sulfate-polyacrylamide (SDS) gel and then transferred onto equilibrated PVDF
membranes. After skimmed milk blocking, the membranes were incubated with primary
antibodies individually with caspase-7, PARP, cleaved caspase-3 (19 kDa and 17 kDa),
phosphorylated Akt (Ser473), p38 MAPK, or p53 (1:1000 dilution). The bound antibodies
were detected with horseradish peroxidase-labelled sheep anti-mouse IgG or horseradish
peroxidase-labelled donkey anti-rabbit IgG (GE Health Care Corp, Piscataway, NJ). The
immunoblots were then developed with enhanced chemiluminescence reagents according to
manufacturer’s recommendations.
Statistical analysis
All data were expressed as mean ± SE. Comparisons among groups were performed by
Student’s t-test. The significance level was set at p < 0.05.
RESULTS
Cisplatin-sensitive (CS) and cisplatin-resistant (CR) human ovarian cancer cells

The cisplatin sensitivity of the two ovarian cancer cell lines was confirmed by a cell-
proliferation assay (MTT) after treating the cells with cisplatin for 24 h (Fig. 1A). The CS and
CR cells exposed to cisplatin (5 μg/ml) for 24 h showed a viability of 42 and 82%, respectively,
confirming their differential sensitivity to cisplatin.
Curcumin inhibits the proliferation of CS and CR cells

The sensitivity of CR ovarian cancer cells to curcumin is unknown. To determine whether
curcumin inhibits the proliferation of CR cell lines, cells were grown in the presence of varying
concentrations of curcumin (0–50 μM) for 48 h and the cell proliferation was measured by
MTT assay. As shown in Figure 1B, curcumin inhibited both CS and CR cell proliferation in
a dose-dependent manner. The IC50 dose of curcumin for proliferation of CS and CR cells was
20 μM. Under similar conditions Chinese hamster ovary (CHO) cells (noncancer) exhibited
an IC50 of 26 μM.
Curcumin induces superoxide generation in ovarian cancer cells

To determine whether curcumin induces superoxide generation in CS and CR cell lines, cells
were grown on chamber slides for 24 h. The cells were then preincubated with DHE (10 μM)
for 1 h, followed by curcumin (10 μM) for 3 h. The nonfluorescent DHE is converted by
superoxide into fluorescent hydroethidine which can be measured by fluorescence microscopy.
Greater fluorescence intensity would indicate more superoxide was present. Both CS and CR
cells showed intense fluorescence (Fig. 2), suggesting the generation of superoxide during
curcumin treatment. CS cells treated with curcumin showed a significantly higher fluorescence
intensity compared to the CR cells (p < 0.01) (Fig. 2). Cells pretreated with N-acetylcysteine
(NAC, 10 mM), an antioxidant, exhibited significantly lower levels of superoxide compared
to the cells not treated with NAC. The superoxide generation in CHO cells treated with
curcumin was significantly lower compared to the cancer cells and NAC completely abolished
the fluorescence intensity.
Weir et al. Page 5
Curcumin increases glutathione levels in ovarian cancer cells

The effect of curcumin on glutathione synthesis in CS and CR cells was determined by
measuring the levels of intracellular GSH by spectrophotometry. Glutathione levels in the
untreated CR cells were significantly higher (p < 0.01) compared to CS cells (Fig. 3). Both CS
and CR cells incubated with curcumin (10 or 50 μM) for 24 h showed increased glutathione
levels. Compared to controls, there were a 214 and 271% increase in glutathione levels in CS
cells and a 168 and 235% increase in CR cells treated with 10 and 50 μM of curcumin,
respectively.
Curcumin causes G2/M Phase cell cycle arrest and induces apoptosis in CR cells
To determine whether curcumin inhibits the cell cycle progression of CR cells, cells were
grown to 70% confluence and the cell cycle distribution was analyzed by flow cytometry after
a 12- and 24-h exposure to curcumin (50 μM). It was observed that the percentage of cells in
G2/M phase with curcumin treatment was 51.5% after 12 h of incubation and decreased to
20.1% after 24 h. The percentage of cells in sub-G0/G1 phase was 3.2% and 35.8% after 12
and 24 h of incubation, respectively. In control cells the percentage of cells in G2/M and Sub-
G0/G1 phase was 20.5% and 1.2% respectively.
Immunoblotting of CR cells treated with curcumin showed increased caspase-7 activity and
cleavages of caspase-3 (19 and 17 kDa), caspase-7 (20 kDa), and PARP (89 kDa) after 12 h
and were even higher after 24 h of incubation (Fig. 5). Quantification of cleaved caspase-3 and
cleaved PARP, done by measuring the relative band intensities, showed that cleaved caspase-3
and PARP levels were significantly higher in CR cells incubated with curcumin for 12 (p <
0.01) and 24 h (p < 0.001) of incubation (Fig. 5B and C). This data indicated that curcumin
induced cell cycle arrest within 12-h of incubation and apoptosis after 24 h of incubation.
Preincubation of CR cells with NAC significantly attenuated the curcumin-induced increase
of cleaved caspase-3 and cleaved PARP. Thus, the data clearly showed that curcumin induced
G2/M phase cell-cycle arrest and apoptosis.
Curcumin inhibits Akt and enhances p38 MAPK activation

To investigate the effect of curcumin on Akt activity in CR cells, we examined the regulation
of Akt phosphorylation by curcumin. The results showed increased phosphorylation of Akt in
CR cells, which was inhibited by curcumin (50 μM) after 12 and 24 h (p < 0.001) of incubation
(Fig. 6). A marked increase in the p38 MAPK and p53 phosphorylation (p < 0.001) was
observed following curcumin exposure for 12 and 24 h (Fig. 6). Pretreatment of the cells with
NAC attenuated the curcumin-induced inhibition of Akt, and activation of p38 MAPK and
p53.
DISCUSSION
The present study demonstrated that curcumin induced cytotoxicity in both cisplatin-sensitive
(CS) and cisplatin-resistant (CR) human ovarian cancer cells. Both cells exhibited similar
responses to curcumin. We also observed that curcumin induced G2/M cell cycle arrest leading
to apoptosis, possibly by down-regulating anti- apoptotic Akt signaling and/or also by
activating the pro-apoptotic p38 MAPK in parallel with the generation of superoxide radicals.
Reactive oxygen species (ROS) such as superoxide radicals are implicated as important
mediators of apoptotic cell death. MAPK is considered as one of the most important signaling
molecules in ROS-mediated apoptosis in cancer cells.37,38 The results of the present study
indicate that curcumin induces superoxide generation in both CS and CR cells but not in CHO
cells. The amount of superoxide generation by curcumin in CR cells was significantly less than
in CS cells. This could be due to the higher levels of endogenous thiol in CR cells as observed
Weir et al. Page 6
in the current study and also in our earlier study.14 On the other hand, curcumin was less
effective in CHO cells wherein the superoxide production was observed to be significantly
less. Curcumin-induced ROS generation has been reported in tumor cells such as rat
histiocytoma (AK-5), human renal carcinoma cell line (Caki cells), human submandibular
gland caracinoma (HSG) was others.39,40 The accumulation of intercellular superoxide may
lead to the disruption of the mitochondrial membrane potential, release of cyctochrome c into
the cytosol, with subsequent activation of the caspase cascade, and apoptosis.41 Therefore, the
curcumin-induced superoxide generation may be critical for the modulation of the apoptotic
signaling pathways.
Our study demonstrated that curcumin inhibited the cisplatin-resistant ovarian cancer cell
proliferation by inducing G2/M cell cycle arrest leading to apoptotic cell death. Recent studies
suggested that caspase-3 plays an important role in several key events causing DNA
fragmentation during apoptosis.42,43 It has been suggested that the cleavage of procaspase-3
is an early event in apoptosis induced by chemotherapeutic agents.44 The activation of
caspase-3 leads to the cleavage of PARP, which serves as a “death substrate”.45 In our study,
cleavages of caspase-7, caspase-3, and PARP were observed in CR cells treated with curcumin.
These results are in agreement with the published reports, which indicate that curcumin induces
apoptosis in cancer cells.18,31 Pretreatment with NAC attenuated the curcumin-induced
cleavage of caspase-3 and 7, and PARP. In colon carcinoma cells, curcumin induced apoptotic
cell death by cell cycle arrest in the S and G2/M phases, whereas in the MCF-7 breast cancer
cell line this occurred at the G2 or M phases.30,46 Previous studies have shown that curcumin
blocks the proliferation of various cancer cells by downregulating the expression of the cyclin
D1 protein.47 In vitro studies have shown that curcumin has multiple biological and
biochemical targets which may be related to its anticancer and antitumor activities.18,48 These
functions include inhibition of PI3 kinase activities and induction of G2/M cell cycle arrest
along with the down-regulation of vascular endothelial growth factor (VEGF).48 Curcumin
induces caspase-3-independent apoptosis in human multidrug-resistant cells such as human
lymphoblastic leukemia cell line and the human colon-carcinoma cell line.49
The PI3-kinase/Akt pathway contributes to the tumor formation by elevating the activity of
the anti-apoptotic action of Akt. Akt inhibits apoptosis through phosphorylation of Bad, GSK3,
and caspase-9 and activation of transcriptional factors such as Forkhead (FOXO1) and NFκB.
50,51 The present study showed increased phosphorylation of Akt in CR cells and curcumin
inhibited Akt phosphorylation. The phosphorylation of Akt is routinely used as readout for the
Akt activation. The inhibition of Akt phosphorylation by curcumin is an important mechanism
of action in CR ovarian cancer cells. Similar results were also observed by others in human
mantle cell lymphoma (MCL).32 Suppression of Akt activation could lead to p53 activation,
which in turn may lead to the activation of pro-apoptotic signaling pathways.52 The regulation
of p53 by Akt is a critical determinant of cisplatin-induced chemoresistance in ovarian cancer

cells.53 In the current study, the observation of an increase in the phosphorylation of p53 in
CR cells treated with curcumin is supported by the PI3K-Akt pathway.
The p38 MAPK pathway is implicated in cancer cell apoptosis and is induced by several
chemotherapeutic drugs.54 Oxidative stress has been reported to play a role in p38 MAPK
activation.38 We found a marked increase in the phosphorylation of p38 MAPK following
curcumin treatment in CR cells. The increased super-oxide production induced by curcumin
subsequently activated p38 MAPK resulting in the activation of caspases, PARP cleavage,
followed by cell death. These findings imply that the ROS trigger is an upstream signal which
initiates the series of apoptotic events induced by curcumin. Pretreatment with NAC attenuated
the curcumin-induced p38 MAPK activation. These results suggest that the activation of the
p38 MAPK pathway plays a causal role in the curcumin-induced apoptosis in CR cells. Thus,
the increased superoxide generation by curcumin may contribute to the enhanced p38 MAPK
Weir et al. Page 7
activity in resistant cells. It was also reported that the loss of the capacity to activate p38 MAPK
in response to cisplatin treatment may be one of the mechanisms of chemoresistance.55 Thus,
activation of p38 MAPK and an increase in the caspase-3 activities appears to contribute to
the proapoptotic effect of curcumin in CR cells. Taken together, our results indicate that
curcumin produces a profound effect on chemoresistant ovarian cancer cell proliferation. The
inhibition of Akt, the activation of p38 MAPK and p53 and the increase in caspase-3 activity
appear to contribute to the proapoptotic effects of curcumin.
The chemoresistance of ovarian cancer was also linked to increased cellular glutathione
content.14,56–58 Depletion of cellular glutathione has been shown to sensitize the resistant
cancer cells to cisplatin and other anticancer drugs.14,59,60 In the present study we observed
that curcumin significantly enhanced the intracellular GSH levels in both CR and CS cells and
but not in CHO cells. However, the anti-proliferative efficacy of curcumin on CR cells was
not significantly different from that of CS cells. These results indicate that in CR cells, curcumin
induces apoptosis through non-glutathione dependent pathways. The exact mechanism of the
enhanced glutathione synthesis in resistant cells is not yet known and warrants further
investigation. In a similar fashion, Piwocka et al, demonstrated the Jurkat cells treated with
curcumin showed increased intracellular GSH levels.49 Curcumin has also been shown to
increase the levels of GSH in kidney cells,61 and rat hepatocytes.62
In summary, the current study demonstrated that curcumin induces G2/M arrest and apoptosis
in cisplatin-resistant human ovarian cancer cells. Further, we showed that curcumin induces
superoxide generation and enhances p38 MAPK and p53 phosphorylation. The increase in Akt
activity in cisplatin-resistant ovarian cancer cells was attenuated by curcumin. Our results
highlight the importance of Akt and p53 signaling pathways as the new targets for the
development of therapeutic strategies against drug-resistant cancer cells, in general, and
cisplatin-resistant cancer cells, in particular. Our results indicate that curcumin may be a
promising compound, especially for the treatment of chemo-resistant human ovarian cancer.
Acknowledgements
We acknowledge the financial support from the NIH grant CA 102264.
References
1. Cancer Facts and Figures. Atlanta: American Cancer Society (ACS); 2005.
2. Smith LH, Morris CR, Yasmeen S, Parikh-Patel A, Cress RD, Romano PS. Ovarian cancer: Can we
make the clinical diagnosis earlier? Cancer 2005;104:1398–407. [PubMed: 16116591]
3. McClay EF, Albright KD, Jones JA, Eastman A, Christen R, Howell SB. Modulation of cisplatin
resistance in human malignant melanoma cells. Cancer Res 1992;52:6790–6. [PubMed: 1458467]
4. McClay EF, Albright KD, Jones JA, Christen RD, Howell SB. Tamoxifen modulation of cisplatin
sensitivity in human malignant melanoma cells. Cancer Res 1993;53:1571–6. [PubMed: 8453625]
5. Cannistra SA. Cancer of the ovary. N Engl J Med 2004;351:2519–29. [PubMed: 15590954]
6. Kelland LR. Emerging drugs for ovarian cancer. Expert Opin Emerg Drugs 2005;10:413–24. [PubMed:
15934876]
7. Arts HJ, Willemse PH, Tinga DJ, de Vries EG, van der Zee AG. Laparoscopic placement of PAP
catheters for intraperitoneal chemotherapy in ovarian carcinoma. Gynecol Oncol 1998;69:32–5.
[PubMed: 9570995]
8. Lee S, Choi EJ, Jin C, Kim DH. Activation of PI3K/Akt pathway by PTEN reduction and PIK3CA
mRNA amplification contributes to cisplatin resistance in an ovarian cancer cell line. Gynecol Oncol
2005;97:26–34. [PubMed: 15790433]
9. Choi KC, Auersperg N, Leung PC. Mitogen-activated protein kinases in normal and (pre)neoplastic
ovarian surface epithelium. Reprod Biol Endocrinol 2003;1:71. [PubMed: 14577832]
Weir et al. Page 8
10. Ikuta K, Takemura K, Kihara M, Nishimura M, Ueda N, Naito S, Lee E, Shimizu E, Yamauchi A.
Overexpression of constitutive signal transducer and activator of transcription 3 mRNA in cisplatin-
resistant human nonsmall cell lung cancer cells. Oncol Rep 2005;13:217–22. [PubMed: 15643501]
11. Jansen BA, Brouwer J, Reedijk J. Glutathione induces cellular resistance against cationic dinuclear
platinum anticancer drugs. J Inorg Biochem 2002;89:197–202. [PubMed: 12062123]
12. Henderson CJ, McLaren AW, Moffat GJ, Bacon EJ, Wolf CR. Pi-class glutathione S-transferase:
Regulation and function. Chem Biol Interact 1998;111–112:69–82.
13. Rudin CM, Yang Z, Schumaker LM, VanderWeele DJ, Newkirk K, Egorin MJ, Zuhowski EG, Cullen
KJ. Inhibition of glutathione synthesis reverses Bcl-2-mediated cisplatin resistance. Cancer Res
2003;63:312–8. [PubMed: 12543781]
14. Bratasz A, Weir NM, Parinandi NL, Zweier JL, Sridhar R, Ignarro LJ, Kuppusamy P. Reversal to
cisplatin sensitivity in recurrent human ovarian cancer cells by NCX-4016, a nitro derivative of
aspirin. Proc Natl Acad Sci USA 2006;103:3914–9. [PubMed: 16497833]
15. Ammon HP, Wahl MA. Pharmacology of curcuma longa. Planta Med 1991;57:1–7. [PubMed:
2062949]
16. Manju V, Nalini N. Chemopreventive efficacy of ginger, a naturally occurring anticarcinogen during
the initiation, post-initiation stages of 1,2 dimethylhydrazine-induced colon cancer. Clin Chim Acta
2005;358:60–7. [PubMed: 16018877]
17. Lodha R, Bagga A. Traditional Indian systems of medicine. Ann Acad Med Singapore 2000;29:37–
41. [PubMed: 10748962]
18. Mukhopadhyay A, Bueso-Ramos C, Chatterjee D, Pantazis P, Aggarwal BB. Curcumin
downregulates cell survival mechanisms in human prostate cancer cell lines. Oncogene
2001;20:7597–609. [PubMed: 11753638]
19. Jagetia GC, Rajanikant GK. Role of curcumin, a naturally occurring phenolic compound of turmeric
in accelerating the repair of excision wound, in mice whole-body exposed to various doses of gamma-
radiation. J Surg Res 2004;120:127–38. [PubMed: 15172199]
20. Dorai T, Aggarwal BB. Role of chemopreventive agents in cancer therapy. Cancer Lett 2004;215:129–
40. [PubMed: 15488631]
21. Sinha R, Anderson DE, McDonald SS, Greenwald P. Cancer risk and diet in India. J Postgrad Med
2003;49:222–8. [PubMed: 14597785]
22. Pal S, Choudhuri T, Chattopadhyay S, Bhattacharya A, Datta GK, Das T, Sa G. Mechanisms of
curcumin-induced apoptosis of Ehrlich’s ascites carcinoma cells. Biochem Biophys Res Commun
2001;288:658–65. [PubMed: 11676493]
23. Rinaldi AL, Morse MA, Fields HW, Rothas DA, Pei P, Rodrigo KA, Renner RJ, Mallery SR.
Curcumin activates the aryl hydrocarbon receptor yet significantly inhibits (−)-benzo(a)pyrene-7R-
trans-7,8-dihydrodiol bioactivation in oral squamous cell carcinoma cells and oral mucosa. Cancer
Res 2002;62:5451–6. [PubMed: 12359752]
24. Choudhuri T, Pal S, Agwarwal ML, Das T, Sa G. Curcumin induces apoptosis in human breast cancer
cells through p53-dependent Bax induction. FEBS Lett 2002;512:334–40. [PubMed: 11852106]
25. Kelloff GJ, Crowell JA, Steele VE, Lubet RA, Malone WA, Boone CW, Kopelovich L, Hawk ET,
Lieberman R, Lawrence JA, Ali I, Viner JL, Sigman CC. Progress in cancer chemoprevention:
Development of diet-derived chemopreventive agents. J Nutr 2000;130:467S–71S. [PubMed:
10721931]
26. Boone CW, Kelloff GJ. Biomarker end-points in cancer chemoprevention trials. IARC Sci Publ
1997:273–80. [PubMed: 9354926]
27. Liu JY, Lin SJ, Lin JK. Inhibitory effects of curcumin on protein kinase C activity induced by 12-O-
tetradecanoyl-phorbol-13-acetate in NIH 3T3 cells. Carcinogenesis 1993;14:857–61. [PubMed:
8504477]
28. Kawamori T, Lubet R, Steele VE, Kelloff GJ, Kaskey RB, Rao CV, Reddy BS. Chemopreventive
effect of curcumin, a naturally occurring anti-inflammatory agent, during the promotion/progression
stages of colon cancer. Cancer Res 1999;59:597–601. [PubMed: 9973206]
29. Aggarwal BB, Kumar A, Bharti AC. Anticancer potential of curcumin: Preclinical and clinical studies.
Anticancer Res 2003;23:363–98. [PubMed: 12680238]
Weir et al. Page 9
30. Chen H, Zhang ZS, Zhang YL, Zhou DY. Curcumin inhibits cell proliferation by interfering with the
cell cycle and inducing apoptosis in colon carcinoma cells. Anticancer Res 1999;19:3675–80.
[PubMed: 10625938]
31. Anto RJ, Mukhopadhyay A, Denning K, Aggarwal BB. Curcumin (diferuloylmethane) induces
apoptosis through activation of caspase-8, BID cleavage and cytochrome c release: Its suppression
by ectopic expression of Bcl-2 and Bcl-xl. Carcinogenesis 2002;23:143–50. [PubMed: 11756235]
32. Shishodia S, Amin HM, Lai R, Aggarwal BB. Curcumin (diferuloylmethane) inhibits constitutive
NF-kappaB activation, induces G1/S arrest, suppresses proliferation, and induces apoptosis in mantle
cell lymphoma. Biochem Pharmacol 2005;70:700–13. [PubMed: 16023083]
33. Rajasingh J, Raikwar HP, Muthian G, Johnson C, Bright JJ. Curcumin induces growth-arrest and
apoptosis in association with the inhibition of constitutively active JAK-STAT pathway in T cell
leukemia. Biochem Biophys Res Commun 2006;340:359–68. [PubMed: 16364242]
34. Bush JA, Cheung KJ Jr, Li G. Curcumin induces apoptosis in human melanoma cells through a Fas
receptor/caspase-8 pathway independent of p53. Exp Cell Res 2001;271:305–14. [PubMed:
11716543]
35. Li L, Aggarwal BB, Shishodia S, Abbruzzese J, Kurzrock R. Nuclear factor-kappaB and IkappaB
kinase are constitutively active in human pancreatic cells, and their down-regulation by curcumin
(diferuloylmethane) is associated with the suppression of proliferation and the induction of apoptosis.
Cancer 2004;101:2351–62. [PubMed: 15476283]
36. Fang J, Lu J, Holmgren A. Thioredoxin reductase is irreversibly modified by curcumin: A novel
molecular mechanism for its anticancer activity. J Biol Chem 2005;280:25284–90. [PubMed:
15879598]
37. Cobb MH. MAP kinases pathways. Prog Biophys Mol Biol 1999;71:479–500. [PubMed: 10354710]
38. Park SJ, Kim IS. The role of p38 MAPK activation in auranofin-induced apoptosis of human
promyelocytic leukaemia HL-60 cells. Br J Pharmacol 2005;146:506–13. [PubMed: 16086031]
39. Bhaumik S, Anjum R, Rangaraj N, Pardhasaradhi BV, Khar A. Curcumin mediated apoptosis in
AK-5 tumor cells involves the production of reactive oxygen intermediates. FEBS Lett
1999;456:311–4. [PubMed: 10456330]
40. Woo JH, Kim YH, Choi YJ, Kim DG, Lee KS, Bae JH, Min do S, Chang JS, Jeong YJ, Lee YH, Park
JW, Kwon TK. Molecular mechanisms of curcumin-induced cytotoxicity: Induction of apoptosis
through generation of reactive oxygen species, down-regulation of Bcl-XL and IAP, the release of
cytochrome c and inhibition of Akt. Carcinogenesis 2003;24:1199–208. [PubMed: 12807727]
41. Singh SV, Srivastava SK, Choi S, Lew KL, Antosiewicz J, Xiao D, Zeng Y, Watkins SC, Johnson
CS, Trump DL, Lee YJ, Xiao H, Herman-Antosiewicz A. Sulforaphane-induced cell death in human
prostate cancer cells is initiated by reactive oxygen species. J Biol Chem 2005;280:19911–24.
[PubMed: 15764812]
42. Blanc C, Deveraux QL, Krajewski S, Janicke RU, Porter AG, Reed JC, Jaggi R, Marti A. Caspase-3
is essential for procaspase-9 processing and cisplatin-induced apoptosis of MCF-7 breast cancer cells.
Cancer Res 2000;60:4386–90. [PubMed: 10969782]
43. Taylor JK, Zhang QQ, Monia BP, Marcusson EG, Dean NM. Inhibition of Bcl-xL expression
sensitizes normal human keratinocytes and epithelial cells to apoptotic stimuli. Oncogene
1999;18:4495–504. [PubMed: 10442640]
44. Henkels KM, Turchi JJ. Cisplatin-induced apoptosis proceeds by caspase-3-dependent and -
independent pathways in cisplatin-resistant and -sensitive human ovarian cancer cell lines. Cancer
Res 1999;59:3077–83. [PubMed: 10397248]
45. Rheaume E, Cohen LY, Uhlmann F, Lazure C, Alam A, Hurwitz J, Sekaly RP, Denis F. The large
subunit of replication factor C is a substrate for caspase-3 in vitro and is cleaved by a caspase-3-like
protease during Fas-mediated apoptosis. Embo J 1997;16:6346–54. [PubMed: 9351817]
46. Simon A, Allais DP, Duroux JL, Basly JP, Durand-Fontanier S, Delage C. Inhibitory effect of
curcuminoids on MCF-7 cell proliferation and structure-activity relationships. Cancer Lett
1998;129:111–6. [PubMed: 9714342]
47. Mukhopadhyay A, Banerjee S, Stafford LJ, Xia C, Liu M, Aggarwal BB. Curcumin-induced
suppression of cell proliferation correlates with down-regulation of cyclin D1 expression and CDK4-
Weir et al. Page 10
mediated retinoblastoma protein phosphorylation. Oncogene 2002;21:8852–61. [PubMed:

12483537]
48. Shao ZM, Shen ZZ, Liu CH, Sartippour MR, Go VL, Heber D, Nguyen M. Curcumin exerts multiple
suppressive effects on human breast carcinoma cells. Int J Cancer 2002;98:234–40. [PubMed:
11857414]
49. Piwocka K, Bielak-Mijewska A, Sikora E. Curcumin induces caspase-3-independent apoptosis in
human multidrug-resistant cells. Ann NY Acad Sci 2002;973:250–4. [PubMed: 12485871]
50. Bharti AC, Donato N, Singh S, Aggarwal BB. Curcumin (diferuloylmethane) down-regulates the
constitutive activation of nuclear factor-kappa B and IkappaBalpha kinase in human multiple
myeloma cells, leading to suppression of proliferation and induction of apoptosis. Blood
2003;101:1053–62. [PubMed: 12393461]
51. Katso R, Okkenhaug K, Ahmadi K, White S, Timms J, Waterfield MD. Cellular function of
phosphoinositide 3-kinases: Implications for development, homeostasis, and cancer. Annu Rev Cell
Dev Biol 2001;17:615–75. [PubMed: 11687500]
52. Fraser M, Leung BM, Yan X, Dan HC, Cheng JQ, Tsang BK. p53 is a determinant of X-linked
inhibitor of apoptosis protein/Akt-mediated chemoresistance in human ovarian cancer cells. Cancer
Res 2003;63:7081–8. [PubMed: 14612499]
53. Yang X, Fraser M, Moll UM, Basak A, Tsang BK. Akt-mediated cisplatin resistance in ovarian cancer:
Modulation of p53 action on caspase-dependent mitochondrial death pathway. Cancer Res
2006;66:3126–36. [PubMed: 16540663]
54. Bulavin DV, Fornace AJ Jr. p38 MAP kinase’s emerging role as a tumor suppressor. Adv Cancer Res
2004;92:95–118. [PubMed: 15530558]

55. Brozovic A, Fritz G, Christmann M, Zisowsky J, Jaehde U, Osmak M, Kaina B. Long-term activation
of SAPK/JNK, p38 kinase and fas-L expression by cisplatin is attenuated in human carcinoma cells
that acquired drug resistance. Int J Cancer 2004;112:974–85. [PubMed: 15386344]
56. Yao KS, Godwin AK, Johnson SW, Ozols RF, O’Dwyer PJ, Hamilton TC. Evidence for altered
regulation of gamma-glutamylcysteine synthetase gene expression among cisplatin-sensitive and
cisplatin-resistant human ovarian cancer cell lines. Cancer Res 1995;55:4367–74. [PubMed:
7671249]
57. Pan B, Yao KS, Monia BP, Dean NM, McKay RA, Hamilton TC, O’Dwyer PJ. Reversal of cisplatin
resistance in human ovarian cancer cell lines by a c-jun antisense oligodeoxy-nucleotide (ISIS
10582): Evidence for the role of transcription factor overexpression in determining resistant
phenotype. Biochem Pharmacol 2002;63:1699–707. [PubMed: 12007573]
58. Okuno S, Sato H, Kuriyama-Matsumura K, Tamba M, Wang H, Sohda S, Hamada H, Yoshikawa H,
Kondo T, Bannai S. Role of cystine transport in intracellular glutathione level and cisplatin resistance
in human ovarian cancer cell lines. Br J Cancer 2003;88:951–6. [PubMed: 12644836]
59. Kachadourian R, Day BJ. Flavonoid-induced glutathione depletion: Potential implications for cancer
treatment. Free Radic Biol Med 2006;41:65–76. [PubMed: 16781454]
60. Benlloch M, Ortega A, Ferrer P, Segarra R, Obrador E, Asensi M, Carretero J, Estrela JM.
Acceleration of glutathione efflux and inhibition of gamma-glutamyltranspeptidase sensitize
metastatic B16 melanoma cells to endothelium-induced cytotoxicity. J Biol Chem 2005;280:6950–

9. [PubMed: 15561710]
61. Venkatesan N, Punithavathi D, Arumugam V. Curcumin prevents adriamycin nephrotoxicity in rats.
Br J Pharmacol 2000;129:231–4. [PubMed: 10694226]
62. White EL, Ross LJ, Schmid SM, Kelloff GJ, Steele VE, Hill DL. Screening of potential cancer
preventing chemicals for induction of glutathione in rat liver cells. Oncol Rep 1998;5:507–12.
[PubMed: 9468590]
Weir et al. Page 11
Figure 1.
Curcumin inhibits the growth of cisplatin-sensitive (CS) and cisplatin-resistant (CR) ovarian
cancer cells. (A) To confirm cisplatin resistance, CS and CR cells were treated with cisplatin
(5 μg/ml) and incubated for 24 h. Viability was determined using the MTT assay. (B) CS, CR
and CHO cells were treated with varying doses of curcumin for 24 h. Cell viability was
determined by MTT assay. Values are expressed as mean ± SE (n = 3). *p<0.01 vs control.
Weir et al. Page 12
Figure 2.
Curcumin induces superoxide generation in CS and CR cells. CS, CR or CHO cells were grown
in chamber slides and were pretreated with dihydroethidium (10 μM) for 30 min and then
exposed to curcumin (10 μM) for 3 h. All experiments were performed with and without NAC
(10 mM). The florescence was monitored using a Nikon fluorescence microscope equipped
with a rhodamine filter. Top, Representative micrographs from triplicate experiments are
shown. Bottom. Fluorescence intensity in curcumin treated CR, CS and CHO cells with and
without NAC are shown. Values are expressed as mean ± SE (n = 3). *p<0.01 vs curcumin.
Weir et al. Page 13
Figure 3.
Curcumin induces glutathione synthesis in CS and CR cells. CS and CR were treated with
curcumin (10 and 50 μM) and incubated for 24 h. Values are expressed as mean ± SE (n = 3).
*p < 0.01 vs CS cells; **p < 0.05 vs control; #p < 0.01 vs control.
Weir et al. Page 14
Figure 4.
Curcumin causes G2/M arrest of CR cells. Flow cytometric analysis of the PI staining of CR
cells after 24 h of growth in the presence of DMSO (control) vehicle or curcumin (50 μM).
Representative values of three experiments are shown. Top: The distribution of total cells in
M1-M4 gate in the flow cytometry plot. Bottom: The percentage of distribution of total cells
in sub-G0-G1, G0/G1, S, and G2/M phase.
Weir et al. Page 15
Figure 5.
Curcumin activates procaspases and PARP degradation in CR cells. CR cells were treated with
curcumin (50 μM) for 12 and 24 h and then Western blot analysis was performed for cleaved
caspase-3, caspase-7, and PARP. Top: Representative blot from 3 independent experiments.
Bottom: Quantification of band intensities. Values are expressed mean ± SE (n=3).
Weir et al. Page 16
Figure 6.
Curcumin inhibits Akt phosphorylation and enhances the p38 MAPK and p53 phosphorylation.
Cells were treated with curcumin (50 μM) for 24 h and then Western blot analysis was
performed for phospho-Akt (Ser473), phospho-p38 MAPK and phospho-p53. Top: A
representative blot from 3 independent experiments is shown. Bottom: Quantification of band
intensities. Values are expressed mean ± SE (n = 3). *p < 0.001 vs control; **p < 0.001 vs
curcumin.

Nihms 16826

Загружено:

Сведения о документе

Исходное описание:

Оригинальное название

Авторское право

Доступные форматы

Поделиться этим документом

Поделиться или встроить документ

Параметры публикации

Этот документ был вам полезен?

Это неприемлемый материал?

Авторское право:

Доступные форматы

Nihms 16826

Загружено:

Авторское право:

Доступные форматы

NIH Public Access

Cancer Biol Ther. 2007 February ; 6(2): 178–184.

Curcumin Induces G2/M Arrest and Apoptosis in Cisplatin-

(NCX-4016, an NO donor) partially restored the cisplatin sensitivity by depleting thiols.14

Curcumin (diferuloylmethane) is a major constituent of turmeric powder which is extracted

MATERIALS AND METHODS

Reagents and culture materials

Enhanced chemiluminescence (ECL) reagents were obtained from Amersham Pharmacia

Cells and culture conditions

Cell proliferation assay

Superoxide determination by DHE fluorescence

Cell cycle analysis by flow cytometry

Cisplatin-sensitive (CS) and cisplatin-resistant (CR) human ovarian cancer cells

Curcumin inhibits the proliferation of CS and CR cells

Curcumin induces superoxide generation in ovarian cancer cells

Curcumin increases glutathione levels in ovarian cancer cells

Curcumin inhibits Akt and enhances p38 MAPK activation

of p53 by Akt is a critical determinant of cisplatin-induced chemoresistance in ovarian cancer

mediated retinoblastoma protein phosphorylation. Oncogene 2002;21:8852–61. [PubMed:

2004;92:95–118. [PubMed: 15530558]

metastatic B16 melanoma cells to endothelium-induced cytotoxicity. J Biol Chem 2005;280:6950–

Вам также может понравиться