Evidence That Curcumin Suppresses the Growth of Malignant Gliomas In Vitro and In Vivo Through Induction of Autophagy: Role of Akt and ERK Signaling Pathways

Molecular Pharmacology Fast Forward. Published on March 29, 2007 as doi:10.1124/mol.106.033167
MOL #33167
Evidence That Curcumin Suppresses the Growth of Malignant Gliomas In Vitro and In
Vivo Through Induction of Autophagy: Role of Akt and ERK Signaling Pathways

Hiroshi Aoki, Yasunari Takada, Seiji Kondo, Raymond Sawaya, Bharat B. Aggarwal, and Yasuko
Kondo

Departments of Neurosurgery (H.A., S.K., R.S., Y.K.) and Experimental Therapeutics (Y.T.,
B.B.A.), The University of Texas M. D. Anderson Cancer Center, Houston, Texas; Department of
Neurosurgery (S.K., R.S.), The Baylor College of Medicine, Houston, Texas; and The Program
in Molecular Pathology (S.K.), The University of Texas Graduate School of Biomedical Sciences
at Houston, Houston, Texas

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Copyright 2007 by the American Society for Pharmacology and Experimental Therapeutics.

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Running Title: Curcumin-induced autophagy in glioma

Abbreviations: mTOR, mammalian target of rapamycin; ERK1/2, extracellular signal–regulated
kinases; NF-κB, nuclear factor κB; p70S6K, p70 ribosomal protein S6 kinase

Manuscript information:
Number of text pages: 34; tables, 0; figures, 6; references, 55.
Number of words in Abstract, 249; Introduction, 785; Discussion, 1,141.

Corresponding authors: Seiji Kondo, Department of Neurosurgery/BSRB1004, The University
of Texas M. D. Anderson Cancer Center, 1515 Holcombe Boulevard, Houston, TX 77030, USA.
Phone: 713-834-6215; Fax: 713-834-6257; E-mail: seikondo@mdanderson.org

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ABSTRACT
Autophagy is a response of cancer cells to various anticancer therapies. It is designated as
programmed cell death type II and characterized by formation of autophagic vacuoles in the
cytoplasm. The Akt/mammalian target of rapamycin (mTOR)/p70S6K and the extracellular
signal–regulated kinases 1/2 (ERK1/2) pathways are two major pathways which regulate
autophagy induced by nutrient starvation. These pathways are also frequently associated with
oncogenesis in a variety of cancer cell types, including malignant gliomas. However, few studies
have examined both of these signal pathways in the context of anticancer therapy–induced
autophagy in cancer cells, and autophagy’s effect on cell death remains unclear. Here, we
examined the anticancer efficacy and mechanisms of curcumin, a natural compound with low
toxicity in normal cells, in U87-MG and U373-MG malignant glioma cells. Curcumin induced
G2/M arrest and non-apoptotic autophagic cell death in both cell types. It inhibited the
Akt/mTOR/p70S6K pathway and activated the ERK1/2 pathway, resulting in induction of
autophagy. Interestingly, activation of the Akt pathway inhibited curcumin-induced autophagy
and cytotoxicity, whereas inhibition of the ERK1/2 pathway inhibited curcumin-induced
autophagy and induced apoptosis, thus resulting in enhanced cytotoxicity. These results imply
that the effect of autophagy on cell death may be pathway specific. In the subcutaneous
xenograft model of U87-MG cells, curcumin inhibited tumor growth significantly (P < 0.05) and
induced autophagy. These results suggest that curcumin has high anticancer efficacy in vitro and
in vivo by inducing autophagy and warrant further investigation toward possible clinical
application in patients with malignant glioma.
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Introduction

Malignant glioma is the most common primary malignant tumor in the brain. In spite of the
combination of surgery, chemotherapy, and radiotherapy, the median survival time of patients
with glioblastoma multiforme, the most malignant type of malignant glioma, is less than one year
from diagnosis (Ohgaki, 2004). Consequently, there is an urgent need to develop new therapeutic
strategies.

Accumulating evidence shows that a natural product, curcumin (diferuloylmethane), has a
potent anticancer effect both in vitro and in vivo on a variety of cancer cell types, such as
leukemia, breast cancer, prostate cancer, and pancreatic cancer (Aggarwal et al., 2003; Shishodia
et al., 2005). However, the efficacy of curcumin for malignant glioma cells in vitro and in vivo is
not yet fully determined. As expected from the fact that curcumin is an active ingredient of the
spice turmeric, it caused no serious toxicity in animal studies (up to 5 g/kg; Wahlstrom and
Blennow, 1978) and it was safely administered to humans without major toxicity in phase I
clinical studies (up to 12 g per day; Sharma et al., 2001; Sharma et al., 2004; Loa et al., 2006).
However, these findings also underscore that we need to overcome the low absorption and
bioavailability of curcumin outside the colon to use it as systemic cancer preventive agent.

Several mechanisms by which curcumin exerts its anticancer effect have been reported. First,
curcumin inhibits a transcription factor, nuclear factor κB (NF-κB), by inhibiting inhibitor of κB
kinase and subsequent IκBα phosphorylation (Singh and Aggarwal, 1995; Bharti et al., 2003;
Aggarwal et al., 2004; Aggarwal et al., 2006). As a result, curcumin down-regulates the
expression of NF-κB–regulated gene products such as Bcl-2, Bcl-XL, cyclin D1, matrix
metalloproteinase-9, cyclooxygenase-2, and interleukin-6, resulting in cell cycle arrest,
suppression of proliferation, and induction of apoptosis (Bharti et al., 2003; Aggarwal et al.,
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2004; Aggarwal et al., 2006; Mukhopadhyay et al., 2002). Second, curcumin inhibits the
Akt/mammalian target of rapamycin (mTOR) pathway and phosphorylation of p70 ribosomal
protein S6 kinase (p70S6K) and eukaryotic initiation factor 4E-binding protein, resulting in
inhibition of proliferation and induction of apoptosis (Aggarwal et al., 2006; Woo et al., 2003;
Bava et al., 2005; Beevers et al., 2006). Other mechanisms of curcumin’s antitumor effect
include down-regulation of transcription factors AP-1 (Nakamura et al., 2002; Prusty and Das,
2005; Tomita et al., 2006) and Egr-1 (Chen et al., 2006).

Recently, autophagy has attracted the interest of scientists in the field of cancer research
because it is designated as programmed cell death type II, while apoptosis is well known as
programmed cell death type I (Bursch et al., 2000). Autophagic cell death is characterized by
numerous autophagic vacuoles in the cytoplasm, while the nucleus remains intact until the late
stage of cell death. In contrast, apoptosis is manifested by DNA condensation and fragmentation.
We have reported that malignant glioma cells are very resistant to apoptosis but that they
undergo autophagy in response to anticancer therapies such as radiation, temozolomide, and
ceramide (Kanzawa et al., 2004; Daido et al., 2004; Daido et al., 2005; Ito et al., 2005).
Autophagy is basically a protein degradation system of the cell’s own lysosomes (Klionsky and
Emr, 2000). It is a process that maintains ATP level and is typically activated on amino acid
deprivation (Meijer and Codogno, 2004; Keleker, 2006). Conversely, amino acids and ATP are
negative regulators of autophagy. As a sensor of amino acids and ATP, mTOR negatively
regulates autophagy through the mTOR/p70S6K pathway by activating this pathway in response
to amino acids and ATP (Blommaart et al., 1995; Shigemitsu et al., 1999). Further, PTEN and
Akt are upstream regulators of the mTOR pathway: PTEN induces autophagy, and Akt inhibits
autophagy (Arico et al., 2001). The Raf-1/MEK1/2/ERK1/2 pathway is another pathway that
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mediates signals stimulated by amino acids: amino acids inhibit this pathway and autophagy.
ERK phosphorylates G α-interacting protein, which accelerates the rate of GTP hydrolysis by the
Gαi3 protein, resulting in induction of autophagy (Ogier-Denis et al., 2000; Pattingre et al., 2003).
Although it is established that the Akt/mTOR/p70S6K pathway and the Raf-1/MEK1/2/ERK1/2
pathway are involved in regulating autophagy, their roles in autophagy in cancer are not yet fully
determined.

In the present study, we investigated the anticancer effect of curcumin on U87-MG and
U373-MG human malignant glioma cells in vitro and in vivo. We found that curcumin efficiently
inhibited growth of these cell types by inducing non-apoptotic autophagic cell death. Further, we
examined the signal pathways of curcumin-induced autophagy and investigated the role of the
pathways in cell death. To the best of our knowledge, this is the first study to demonstrate that
curcumin induces autophagy, which is regulated by simultaneous inhibition of the
Akt/mTOR/p70S6K pathway and stimulation of the ERK1/2 pathway.


Materials and Methods
Reagents. Curcumin with a purity greater than 95% was kindly supplied by Sabinsa
Corporation (Piscataway, NJ) and dissolved in DMSO (Sigma) to produce a 100 mM stock
solution. Acridine orange was purchased from Polysciences (Warrington, PA). 3-Methyladenine
(3-MA), a phosphatidylinositol 3-phosphate kinase (PI3K) inhibitor, was purchased from Sigma.
PD98059, a mitogen-activated protein kinase/extracellular signal-regulated kinase kinase 1
(MEK1) inhibitor, was purchased from Cell Signaling Technology (Beverly, MA). Paclitaxel
(Taxol) was purchased from Bristol-Myers Squibb (Princeton, NJ). A recombinant full-length
human active Akt1 protein (rAkt1) was purchased from Upstate (Temecula, CA).
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Cell Culture. U87-MG and U373-MG human malignant glioma cells with PTEN mutation
and KBM-5 human leukemia cells were purchased from the American Type Culture Collection
(Manassas, VA). Cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM)
supplemented with 10–15% fetal bovine serum (Invitrogen, Carlsbad, CA), 100 U/ml of
penicillin (Invitrogen), and 2.5 µg/ml of antimycotic (Fungizone; Invitrogen) at 37°C in 5% CO2.
Cell Viability Assay. The cytotoxic effect of curcumin was determined by using the cell
proliferation reagent WST-1 (Roche Applied Science, Indianapolis, IN), as described previously
(Ito et al., 2006). Briefly, U87-MG and U373-MG cells were seeded at 3 × 103 cells/well in 96-
well flat-bottomed plates and incubated at 37°C overnight. After cells were treated with 0, 10, 30,
50, 70, or 90 µM curcumin for 72 hours, they were exposed to 10 µl of the WST-1 reagent for 1
hour at 37°C. The absorbance at 450 nm was measured using a microplate reader. The viability
of untreated cells was considered to be 100%.
Cell Cycle Analysis. Tumor cells treated with curcumin (0, 20, and 40 µM) for 72 hours
were trypsinized, fixed with ice cold 70% ethanol, stained with propidium iodide by using a
cellular DNA flow cytometric analysis reagent set (Roche), and analyzed for DNA content by
FACScan (Becton Dickinson, San Jose, CA). Data were analyzed by Cell Quest software
(Becton Dickinson). At least 100,000 cells were analyzed for each sample. Paclitaxel (5 nM) was
used as a positive control to induce apoptosis (Kondo and Kondo, 2006).
Apoptosis Detection Assay. Tumor cells were seeded on Lab-Tek chamber slides (Nunc,
Rochester, NY) and incubated overnight, and then were treated with 40 µM curcumin for 72
hours and stained with the terminal deoxynucleotidyl transferase–mediated dUTP nick-end
labeling (TUNEL) technique using an ApopTag apoptosis detection kit (Chemicon) as described
previously (Takeuchi et al., 2005). Two hundred cells were counted and scored for the incidence
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of positive staining under a microscope. Paclitaxel (5 nM) was used as a positive control to
induce apoptosis.
Clonogenic Assay. Tumor cells were diluted serially and seeded into the 6-well plates in
triplicate per data point. Twenty-four hours after seeding, cells were treated with different
concentrations of curcumin as indicated. Two weeks after treatment, cells were fixed and stained
with 0.5 % crystal violet (Sigma) in methanol for 5 minutes. And then colonies consisting 50 or
more cells were counted.
Electron Microscopy. To detect the induction of autophagy morphologically in curcumin-
treated tumor cells, we performed ultrastructural analysis. Cells were grown on glass coverslips,
treated with 40 µM curcumin for 48 hours, and then fixed with a solution containing 3%
glutaraldehyde plus 2% paraformaldehyde in 0.1 M cacodylate buffer, pH 7.3, for 1 hour. After
fixation, the samples were postfixed in 1% OsO4 in the same buffer for 1 hour and then subjected
to electron microscopic analysis. Representative areas were chosen for ultrathin sectioning and
viewed with a JEM 1010 transmission electron microscope (JEOL, Peabody, MA) at an
accelerating voltage of 80 kV. Digital images were obtained with an AMT imaging system
(Advanced Microscopy Techniques, Danvers, MA).
Detection and Quantification of Acidic Vesicular Organelles with Acridine Orange
staining. Autophagy is the process of sequestering cytoplasmic proteins into the lytic component
and is characterized by the formation and promotion of acidic vesicular organelles (AVOs), as
described previously (Paglin et al., 2001). To detect the development of AVOs, we treated cells
with 20 and 40 µM curcumin for 72 hours and then performed vital staining with acridine orange.
To quantify the development of AVOs, the cells were stained with acridine orange (1 µg/ml) for
15 minutes, removed from the plate with trypsin-EDTA (Invitrogen), and analyzed using a
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FACScan flow cytometer and CellQuest software. To inhibit autophagy, 2.0 mM 3-MA was
added 24 hours after addition of curcumin.
GFP-LC3 Dot Assay. The green fluorescent protein (GFP)-tagged microtubule-associated
protein 1 light chain 3 (LC3) expression vector was kindly provided by Dr. Noboru Mizushima
(Tokyo Medical and Dental University, Tokyo, Japan). LC3 is recruited to the autophagosomal
membrane during autophagy (Kabeya et al., 2000). Therefore, GFP-tagged LC3-expressing cells
have been used to demonstrate induction of autophagy (Kanzawa et al., 2004; Kabeya et al.,
2000; Mizushima, 2004). GFP-LC3 cells present a diffuse distribution under control conditions,
whereas a punctate pattern of GFP-LC3 expression (GFP-LC3 dots) is induced by autophagy.
Cells were transiently transfected with the GFP-LC3 vector using Fugene 6 transfection reagent
(Roche). After overnight culture, cells were treated with 20 and 40 µM curcumin for 72 hours,
fixed with 4% paraformaldehyde, and examined under a fluorescence microscope. To quantify
autophagic cells after curcumin treatment, we counted the number of autophagic cells
demonstrating GFP-LC3 dots (≥ 10 dots per cell) among 200 GFP-positive cells.
Western Blotting. Soluble proteins were isolated from untreated and curcumin-treated cells.
For the detection of LC3, poly(ADP-ribose) polymerase (PARP), and NF-κB p65, culture
medium with 10% FBS was used. For the detection of signal pathway molecules phospho-Akt,
phospho-p70S6K, and phospho-ERK, culture medium with low serum (0.5% FBS) was used for
up to 24 hours to exclude the effects of growth factors contained in the serum. Equal amounts of
protein were separated by 10% or 15% SDS-PAGE gel (Bio-Rad, Richmond, CA) and
transferred to a Hybond-P membrane (GE Healthcare, USA). The membranes were treated with
primary antibodies overnight at 4°C and incubated for 1 hour with a horseradish peroxidase–
conjugated anti-mouse or anti-rabbit secondary antibody (1:3,000 dilution; GE Healthcare) at
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room temperature for 1 hour. Bound antibody complexes were detected using an enhanced
chemiluminescence reagent (GE Healthcare) according to the manufacturer’s instructions. Anti-
LC3 antibody against a synthetic peptide corresponding to the N-terminal 14 amino acids of
isoform B LC3 and an additional cysteine (PSEKTFKQRRTFEQC) was prepared by
immunization of rabbit and was affinity purified on an immobilized peptide-Sepharose column
(Covance, Denver, PA). We purchased anti–β-actin (Sigma), anti–phospho-Akt at Ser473, anti-
total Akt, anti–phospho-p70S6K at Thr389, anti-total p70S6K, anti–phospho-ERK1/2 at
Thr202/Tyr204, anti-total ERK1/2, anti–phospho-PP1α at Thr320, anti-total PP1α, and anti-PARP
antibodies from Cell Signaling Technology. Anti–phospho-PP2A at Thr307 and anti-total PP2A
antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). To inhibit MEK1,
25 µM PD98059 was added 1 hour before curcumin treatment.
PI3K Activity Assay. PI3K activity was measured using PI 3-Kinase ELISA kit (Echelon
Bioscience, Inc., Salt Lake City, UT) according to the manufacture’s instructions. This kit
evaluates PI3K activity to detect the conversion of PI(4,5)P2 into PI(3,4,5)P3. Briefly, cell
culture and treatments were the same as described above. The cells were rinsed three times with
ice cold Buffer A (137 mM NaCl, 20 mM Tris-Hcl, pH 7.4, 1 mM CaCl2, 1 mM MgCl2, and 0.1
mM sodium orthovanadate) and harvested with ice cold Lysis Buffer (Buffer A plus 1 % NP-40
and 1 mM PMSF). The cellular proteins were extracted by centrifugation. PI3K was isolated
from equal amounts (100 µg) of the cellular protein by immunoprecipitation using anti-PI3K
antibody (Upstate Biotechnology, Lake Placid, NY). The immuno complexes bound onto protein
A-agarose beads were incubated in the reaction buffer containing PI(4,5)P2 substrate and ATP,
and the kinase reaction was stopped by pelleting the beads by centrifugation. The reaction
mixtures were used for the following detection reaction. The absorbance of final solution was
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