Attenuation of proteolysis and muscle wasting by curcumin c3 complex
in MAC16 colon tumour-bearing mice
Rafat A. Siddiqui1*, Samira Hassan1, Kevin A. Harvey1, Tamkeen Rasool1, Tapas Das2, Pradip Mukerji2
and Stephen DeMichele2
1Methodist Research Institute, Clarian Health Partners, 1800 N. Capitol Avenue, Indianapolis, IN 46202, USA
2Research and Development, Abbott Nutrition, Abbott Laboratories, Columbus, OH 43215, USA
(Received 17 October 2008 – Revised 18 March 2009 – Accepted 19 March 2009 – First published online 27 April 2009)
Muscle wasting or cachexia is caused by accelerated muscle protein breakdown via the ubiquitin–proteasome complex. We investigated the effect
of curcumin c3 complex (curcumin c3) on attenuation of muscle proteolysis using in vitro and in vivo models. Our in vitro data indicate that
curcumin c3 as low as 0·50mg/ml was very effective in significantly inhibiting (30%; P,0·05) tyrosine release from human skeletal muscle
cells, which reached a maximum level of inhibition of 60% (P,0·05) at 2·5mg/ml. Curcumin c3 at 2·5mg/ml also inhibited chymotrypsin-
like 20S proteasome activity in these cells by 25% (P,0·05). For in vivo studies, we induced progressive muscle wasting in mice by implanting
the MAC16 colon tumour. The in vivo data indicate that low doses of curcumin c3 (100mg/kg body weight) was able to prevent weight loss in
mice bearing MAC16 tumours whereas higher doses of curcumin c3 (250mg/kg body weight) resulted in approximately 25% (P,0·05) weight
gain as compared with the placebo-treated animals. Additionally, the effect of curcumin c3 on preventing and/or reversing cachexia was also
evident by gains in the weight of the gastrocnemius muscle (30–58%; P,0·05) and with the increased size of the muscle fibres (30–65%;
P,0·05). Furthermore, curcumin inhibited proteasome complex activity and variably reduced expression of muscle-specific ubiquitin ligases: atro-
gin-1/muscle atrophy F-box (MAFbx) and muscle RING finger 1 (MURF-1). In conclusion, oral curcumin c3 results in the prevention and reversal
of weight loss. The data imply that curcumin c3 may be an effective adjuvant therapy against cachexia.
MAC16 tumours: Cachexia: Curcumin: Proteasomes: Muscle wasting
Cachexia is defined as the progressive wasting of body tissues
that primarily affects muscle and adipose tissue(1–3). Patients
suffering from sepsis, trauma, AIDS and many types of
cancer exhibit cachexia(4–10). All cancer patients exhibit
some degree of cachexia, and it is one of the most important
factors leading to early morbidity and mortality, accounting
for up to 30% of all deaths(11). It is particularly more pro-
nounced in pancreatic and head–neck cancers(6,12–14). Studies
during the last 10 years have concluded that muscle wasting is
primarily caused by accelerated muscle protein breakdown
via the ubiquitin–proteasome complex(15). Expression in
muscle of two ubiquitin ligases, namely mouse atrophy gene-1
(atrogin-1) (also described as the muscle atrophy F-box;
MAFbx) and the muscle RING finger 1 (MURF-1)(16,17), is
up-regulated in various animal models of muscle atrophy,
including fasting, cancer, sepsis, disuse, denervation, diabetes
and uraemia(16–20). Similarly, muscle wasting situations in
humans, including immobilisation(21), acute quadriplegic
myopathy and neurogenic atrophy(22), are also accompanied
with up-regulated MAFbx/atrogin-1 and MURF-1 expression.
Various inflammatory cytokines, including TNFa, inter-
feron g, IL-6, leukaemia inhibitory factor, and mediators,
including proteolysis-inducing factor and lipid mobilising
factor, are known to play a role in the development of
cachexia(23). Currently, therapies aimed at neutralising these
cytokines or mediators have had only limited success(24).
Furthermore, anorexia is often accompanied by cachexia;
however, refeeding a balanced diet does not reverse the pro-
gression of cachexia(25). Recently, attempts have been made
to supplement diets with nutrients that specifically inhibit
muscle proteolysis(26). One such promising supplement is
diene-3,5-dione), which is present in turmeric. Curcumin has
anti-inflammatory, antioxidant, anticarcinogenic, antidiabeto-
genic, antibacterial, antiviral and free radical-scavenging
properties(27–31). The pharmacology and putative anticancer
properties of curcumin have been extensively reviewed(32).
Recent studies indicate that curcumin may also possess
antiproteolytic properties. For example, curcumin is reported
to inhibit proteasome activity in HeLa cells(33), as well as
attenuate the proteolysis-inducing factor-induced increase in
proteasome activity in the muscle tissues of tumour-bearing
mice(34). The intraperitoneal administration of curcumin
daily for four consecutive days before a lipopolysaccharide
*Corresponding author: Dr Rafat A. Siddiqui, fax þ1 317 962 6941, email email@example.com
Abbreviations: LPS, lipopolysacchride; MAFbx, muscle atrophy F-box; MURF-1, muscle RING finger 1; SkBM, Skeletal Muscle Cell Basal Medium; Tris, 2-amino-
British Journal of Nutrition (2009), 102, 967–975
q The Authors 2009
British Journal of Nutrition
(LPS) injection in mice blunted LPS stimulation of atrogin-1/
MAFbx mRNA expression in gastrocnemius and extensor
digitorum longus muscle of mice(35). Curcumin has been
shown to increase the rate and extent of muscle regeneration
after trauma(36). In contrast to these studies, intraperitoneal
administration of curcumin (20mg/kg body weight) has been
ineffective in preventing muscle wasting or changes in the
body weight of rats bearing the highly cachectic Yoshida
AH-130 ascites hepatoma(37). Similarly, curcumin treatment
by mouth at higher dose levels (150 and 300mg/kg body
weight) was shown to be ineffective in preventing loss of
body weight in mice bearing the MAC16 colon tumour(34).
It is, therefore, not clear why a compound that has potential
inhibitory activities against protein degradation in vitro
failed to reverse cachexia in experimental models. Low curcu-
min absorption and hence bioavailability have been suggested
as the primary reasons for the failure of curcumin(34,38). We
hypothesised that this failure of curcumin in those studies
was due to using an animal model that exhibited a drastic
body-weight loss (20% over 5d) as well as short treatment
duration (4–5d). To test our hypothesis, we used a standar-
dised patented curcumin extract, and reinvestigated its effect
on proteolysis in human skeletal muscle cells, and also treated
cachectic animals that were gradually losing body weight
(20% over 21d) for a longer duration (21d).
Proliferating human skeletal myoblast cells (Cambrex Bio
Science, Walkersville, MA, USA) were cultured in Clone-
ticswSkeletal Muscle Cell Basal Medium (SkBMw; Cambrex
Bio Science) supplemented with bovine serum albumin,
bovine fetuin, insulin, dexamethasone, recombinant human
epidermal growth factor and gentamycin sulfate with ampho-
tericin B (all supplied with SkBMwas SingleQuotsw), accord-
ing to the manufacturer’s instructions. Cells were not
differentiated into myotubules under these conditions. Cells
were subcultured when they were approximately 70%
Validation of curcumin c3 complex
The present study was performed using curcumin c3 complex,
a standardised preparation (Sabinsa, Piscataway, NJ, USA)
whichhas been usedextensively
trials(39–41). For validation, the composition of curcumin c3
complex was analysed by a reversed-phase HPLC method
using an Agilent 1100 HPLC system (Agilent Technologies,
Inc., Santa Clara, CA, USA) equipped with an Agilent 1100
diode-array detector, an Agilent 1100 autosampler system
(4·6 £ 150mm; 5mm). The mobile phase consisted of A
(15% acetonitrile in 0·05 M-KH2PO4; pH 2·9) and B (80%
acetonitrile in distilled water) solvents as follows: 0% solvent
B from 0–5min; 0–80% solvent B from 5–30min (linear
gradient); 100% solvent B from 30·1–35min; 0% solvent B
from 35·1–45min (end) at a flow rate of 0·5ml/min. The
detection wavelengths were 260 and 428nm. Chromato-
graphic peaks were identified by comparing retention times
of samples with those of standards (curcumin, bisdemethoxy-
curcumin, demethoxycurcumin) as described(42).
Cytotoxic effects of curcumin
Cells (1 £ 104per well) were seeded in a ninety-six-well plate
overnight and then treated with varying concentrations of cur-
cumin c3 in serum-free medium for 24h. Curcumin c3 was
dissolved in dimethyl sulfoxide (5mg/ml) as a stock solution.
A sample of curcumin c3 was diluted in media before treat-
ment. The final concentration of dimethyl sulfoxide was
kept at 0·1%. The control cells were treated with vehicle
only (0·1% dimethyl sulfoxide). The effect of the curcumin
c3 on skeletal muscle cell viability was determined with a
water-soluble tetrazolium salt-1 (2-(4-iodophenyl)-3-(4-nitro-
salt) assay in accordance with the manufacturer’s instructions
(Roche Biosciences, Indianapolis, IN, USA). This assay
is based on mitochondrial dehydrogenase activity, which is
present only in the respirating viable cells.
In vitro protein degradation
Protein degradation in response to serum starvation was
assayed as described(43). Briefly, skeletal muscle cells were
plated in twenty-four-well tissue culture plates for 24h, then
rinsed with serum-free SkBMwculture media, and finally
labelled under serum-free conditions with L-[3, 53H]tyrosine
(1905·3GBq (51·50Ci)/mmol; 0·036996MBq (1mCi)/ml in
each well) for another 24h. Labelled monolayers were
washed three times with serum–free SkBMwcontaining
50mM–cycloheximide and 2mM–unlabelled tyrosine and
then incubated in the same media (2ml) for 48h in the pre-
sence or absence of curcumin c3. A sample of the media
was then removed (750ml) for determining radioactive tyro-
sine release, mixed with 5ml of ScintiVerse (Fisher Scientific,
Hanover Park, IL, USA) and the radioactivity was quantified
using a Beckman L6000 liquid scintillation counter (Beckman
Coulter, Inc., Fullerton, CA, USA). Inhibition of tyrosine
release in the media by curcumin c3 was calculated from tyro-
sine release in the absence of curcumin (control, 100%) after
correcting for subtraction of background counts.
Animal model of cachexia
The murine MAC16 colon tumour model for inducing
cachexia in mice was established as previously described(44).
Murine MAC16 tumour cells originally derived in Dr Michael
Tisdale’s laboratory (Aston University, Birmingham, UK)
were kindly provided by Dr Constance Monitto (John Hopkins
Hospital, Baltimore, MD, USA). Cells were initially main-
tained in culture in Roswell Park Memorial Institute (RPMI)
1640 medium with L-glutamine (GIBCO BRL; Life Technol-
ogies, Rockville, MD, USA) containing 12% fetal bovine
serum (HyClone, Logan, UT, USA) and penicillin–streptomy-
cin (100U/ml and 100mg/ml, respectively) in a humidified
atmosphere with 5% CO2 at 378C. For tumour induction,
200ml of MAC16 cells (5 £ 106/ml in PBS) were injected
subcutaneously in the lower back of Hsd:Athymic nude–nu
male mice (aged 6–7 weeks, average body weight 26·71
(SD 1·31) g; Harlan Laboratories, Indianapolis, IN, USA).
R. A. Siddiqui et al. 968
British Journal of Nutrition
Once the tumour was palpable the body weight of the animals
and progression of tumour growth were recorded every day
post–tumour implantation (PI) using a digital caliper (Fisher
Scientific, Pittsburgh, PA, USA). Animals that exhibited a
loss of 5–7% of initial body weight (10–12d PI) were ran-
domised into treatment groups (five animals per group).
Mice were orally administered daily with placebo vehicle
(200ml olive oil) or 100mg/kg body weight or 250mg/kg
body weight of curcumin c3 (in 200ml olive oil) as
described(45). Animals were given a standard laboratory
non–purified diet (LabDiet, catalogue no. 5001; Ted’s Feed,
Indianapolis, IN, USA) and water ad libitum. The diet consists
of 24% proteins, 10·7% fats, 48% carbohydrates and 5%
fibres. Upon completion of the study, mice were euthanised
by inhalation of the anaesthetic gas isoflurane. Mice were
skinned and tumours removed to measure the carcass body
weight. Hindquarters were removed and weighed. Gastrocne-
mius muscles from both legs were removed, weighed and
quickly frozen in liquid N2. Thigh muscles were used for
tissue sections. Tissue specimens were stored at 2808C for
biochemical analysis. The protocol for these studies was
approved by the Methodist Research Institute’s Animal
Research Committee and strictly followed the Guide for the
Care and Use of Laboratory Animals (National Institutes of
Health publication no. 85-23, revised 1996).
Transverse serial sections of quadriceps muscle (10mm) were
prepared using a cryostat (Leica CM1900; Leica Micro-
systems, Bannockburn, IL, USA). The sections were stained
using haematoxylin and eosin staining (Sigma Chemical Co.,
St Louis, MO, USA). Images of rectus femoris muscle sec-
tions were recorded using a digital camera (mounted on a
microscope) as described(46). The surface area of individual
muscle bundles (representing mixed fibre types) were
measured using ImagePro software (Cybernetics, Silver
Spring, MD, USA).
The effect of curcumin c3 on proteasome activity was assayed
in both muscle extracts of MAC16 tumour-bearing mice as
well as in serum-starved human skeletal muscle cells. Gastro-
cnemius muscles of mice were homogenised using a
polytron homogeniser in 20mM-2-amino-2-hydroxymethyl-
propane-1,3-diol (Tris)-HCl (pH 7·5) containing 2mM-ATP,
5mM-MgCl2 and 1mM-dithiothreitol. The homogenate was
centrifuged at 800g and the supernatant fraction was used
for determining the chymotrypsin-like activity of the 20S
proteasome using a kit (20S Proteasome Activity kit; Boston
Biochem, Cambridge, MA, USA). The activities were adjusted
for protein concentrations. Other protease-like activities of the
proteasome complex were not determined.
Western blot analysis
Muscle tissues from animals were homogenised in a homogen-
ising buffer (0·25 M-sucrose, 50mM-HEPES (pH 7·4), 2mM-
ethylene glycol tetraacetic acid) using a polytron homogeniser.
The homogenate was solubilised in 1:1 ratios with lysis buffer
2mM-Na3VO4, 10% glycerol, 1% nonidet P-40, 2mM-phe-
nylmethanesulfonylfluoride, leupeptin (1mg/ml), aprotinin
(0·15 units/ml) and 2·5mM-diisofluorophosphate) for 10min
on ice. The detergent-solubilised extracts were centrifuged
to remove insoluble matter. After evaluating the protein con-
tent using a bicinchoninic acid (BCA) Protein Assay Kit
(Pierce, Rockford, IL, USA), 15mg of protein solubilised in
Laemmli sample loading buffer was loaded onto each lane
of a 4–12% gradient SDS–polyacrylamide gel. Proteins
were electrophoretically separated and transferred onto nitro-
cellulose membranes (Millipore Corporation, Bedford, MA,
USA) for immuno-Western blot analysis. Blots were then
incubated with anti-MAFbx (Oncogene Research Products,
Calbiochem, San Diego, CA, USA) and anti-MURF-1 (Onco-
gene Research Products) primary antibodies (1:1000 dilution
in Tris-buffered saline with Tween-20) according to the man-
ufacturer’s specifications and proteins were detected using a
peroxidase-conjugated secondary antibody (1:5000 dilution
in Tris-buffered saline with Tween-20 containing 1% bovine
serum albumin) with an enhanced chemiluminescence (ECL)
system (Amersham Pharmacia Biotechnology, Piscataway,
NJ, USA). For reprobing, membranes were stripped in
buffer consisting of 62·5mM-Tris-HCl (pH 6·8), 2% SDS
and 100mM-b-mercaptoethanol for 30min at 508C followed
by six washes in Tris-buffered saline (pH 7·4) with 0·1%
Tween 20. To verify an equal distribution of protein loading,
blots were reprobed with a peroxidase-conjugated glyceralde-
hyde 3-phosphate dehydrogenase antibody (1:1000 dilution in
Tris-buffered saline with Tween-20; Santa Cruz Biotechnol-
ogy, Santa Cruz, CA, USA). The resolved proteins were quan-
(MM2000; Kodak, Rochester, NY, USA).
a Kodak ImageStation
One-way ANOVA was used for overall comparisons across all
treatment groups. Post hoc pairwise comparisons were per-
formed using Tukey’s multiple-comparison test. Statistical
analysis was completed using Minitab 14.2 (Minitab Inc.,
State College, PA, USA). Data were summarised by treatment
group using mean and standard error. The Student’s t test was
also used (as mentioned elsewhere) to determine differences
between individual groups compared with control. A value
of P,0·05 was considered statistically significant.
Curcumin c3 composition
We tested the purity of curcumin c3 complex and found it to
contain 73% curcumin, 22% desmethoxycurcumin and 4%
bis-desmethoxycurcumin as reported by the manufacturer.
Treatment of curcumin c3 at 100mg/kg body weight therefore
contained 73mg curcumin, 22mg desmethoxycurcumin and
4mg bis-desmethoxycurcumin/kg body weight, whereas treat-
ments with 250mg curcumin c3/kg body weight contained
182·5mg curcumin, 55mg desmethoxycurcumin and 10mg
bis-desmethoxycurcumin/kg body weight.
Attenuation of proteolysis by curcumin969
British Journal of Nutrition
Curcumin c3 inhibits protein breakdown and chymotrysin-like
20S proteasome activity in skeletal muscle cells
In an initial screen, we investigated cytotoxic concentrations
of curcumin c3 for human skeletal muscle cells. Curcumin
c3 was well tolerated up to a concentration of 2·5mg/ml. Con-
centrations over 2·5mg/ml appeared to be toxic, causing total
cell death at 5mg/ml (data not shown). The subsequent exper-
iments were therefore performed under non-toxic concen-
trations. Data shown in Fig. 1(a) demonstrate that curcumin
c3 has a dose-dependent effect on protein degradation as
assayed by tyrosine release during serum starvation. It is
clear from the data that a curcumin c3 concentration as low
as 0·50mg/ml was very effective in significantly inhibiting
(30%; P,0·05) tyrosine release, which reached a maximum
level of inhibition of (60%; P,0·05) at 2·5mg/ml. Consistent
with this result, our data in Fig. 1(b) demonstrate that
curcumin c3 at 2·5mg/ml was able to inhibit chymotrysin-
like proteasome 20S activity by 25% (P,0·05).
Curcumin c3 treatment prevents body-weight loss
Data presented in Fig. 2 indicate that placebo-treated tumour-
bearing animals progressively lost body weight, reaching a
total loss of approximately 18–20% at day 21. When treated
at a lower dose of curcumin c3 (100mg/kg body weight),
MAC16-tumour-bearing animals effectively maintained their
initial loss of 4–7% of body weight (P,0·05 compared
with placebo). However, tumour-bearing animals treated
with a higher dose of curcumin c3 (250mg/kg body weight)
initially resisted any loss of body weight during the first
10d treatment and then started gaining weight and were
able to increase their weight by 8–10% from their initial
body weight by day 21 (P,0·05) and by 25% (P,0·05)
These animals achieved comparable body-weight gains to
those of non-tumour-bearing placebo-treated animals (Fig. 2).
Subtraction of the tumour’s weight from the animal’s weight
at the end of the study (day 21 post-treatment) indicated that
low and high doses of curcumin c3 treatment caused a net
increase in body weight by 24% (P,0·05) and 35%
(P,0·05), respectively, compared with that of placebo-treated
animals (Table 1). The animals in all three groups did not
exhibit significant differences in tumour weights at day 21
(Table 1). Moreover, the animals did not exhibit significant
differences in average daily food intake before and after
tumour implantation (Table 1).
Curcumin improves muscle characteristics
We next examined whether the increase in body weight in
tumour-bearing animals on curcumin c3 treatment was due
Fig. 1. Inhibition of proteolysis (a) and chymotrysin-like 20S proteasome
activity (b) in muscle cells by curcumin c3. (a) Inhibition of proteolysis was
determined by monitoring the release of radioactive tyrosine from pre-
labelled human skeletal muscle cells in the presence or absence of curcumin
(control, 100%). The radioactivity was quantified using a Beckman L6000
liquid scintillation counter (Beckman Coulter, Inc., Fullerton, CA, USA). (b)
20S chymotrysin-like proteasome activity was determined in the supernatant
fraction of cell homogenate using a kit (20S Proteasome Activity Kit; Boston
Biochem, Cambridge, MA, USA). The activities were adjusted for protein
concentrations. Values are the means for at least three experiments, with
standard errors represented by vertical bars. * Mean value was significantly
different from that of the control treatment (P,0·05).
Fig. 2. Effect of curcumin c3 on body weight of MAC-16 tumour-bearing
mice. Animal body weights were recorded every day post-tumour implan-
tation (PI). Animals (n 5) that exhibited a loss of 5–7% of initial body weight
(10–12d PI) were randomised into treatment groups. Mice were orally admi-
nistered daily with placebo vehicle (200ml olive oil; –X–) or 100mg (–P–)
or 250mg (–B–) curcumin c3/kg body weight (in 200ml olive oil). Non-
tumour bearing mice (n 3) of comparable weights were also treated with
vehicle (200ml olive oil) for comparison (–V–). Values are means, with stan-
dard errors represented by vertical bars. The results were analysed using
different (P,0·001). Using Tukey’s multiple-comparison test, significant
differences were found between groups (P,0·05).
a,b,cMean values at day 21 with unlike letters were significantly
R. A. Siddiqui et al.970
British Journal of Nutrition
to improvements in the animals’ muscle characteristics.
Weights of gastrocnemius muscle were increased by 30%
(P,0·05) and 58% (P,0·05) in low- and high-curcumin
c3-treated animals, respectively, compared with those of pla-
cebo-treated animals (Table 1). Consistent with these obser-
vations, muscle fibre size was also increased, as depicted in
Fig. 3(a). Quantification of these muscle fibres indicated that
their size increased by 30% (P,0·05) and 65% (P,0·05)
in low- and high-curcumin c3-treated animals, respectively,
compared with that of placebo-treated animals (Fig. 3(b)).
The weight of hindquarters was statistically not different in
animals treated with low (P,0·12) and high (P,0·06) curcu-
min c3, compared with placebo-treated animals (Table 1).
Curcumin c3 inhibits muscle proteolysis
We further examined whether improvements in muscle
characteristics after curcumin c3 treatment was a result of cur-
cumin c3’s effect on the proteasome pathway. Curcumin c3
treatment resulted in the inhibition of chymotrypsin-like pro-
teasome 20S activity by 22–25% (P,0·05) as compared
with placebo (Fig. 4(a)). However, there was no significant
difference between low v. high curcumin c3 treatment. We
further examined the expression of atrogin-1/MAFbx and
(Fig. 4(b)). Expression of atrogin-1/MAFbx was reduced by
20–25% (P¼0·11) in isolated gastrocnemius muscle on cur-
cumin c3 treatment compared with that of controls (placebo
treatment); however, expression of MURF-1 was reduced by
(Fig. 4(c)). It is interesting to note that a significant difference
(P,0·05) in the inhibition of MURF-1 expression was
observed when animals treated with high doses of curcumin
were directly compared with the control group using Student’s
on treatmentwith curcuminc3
Our data indicate that a low dosage of curcumin c3 (100mg/
kg body weight) was able to prevent weight loss in mice bear-
ing cachexia-inducing MAC16 tumours whereas a higher
dosage at 250mg/kg body weight resulted in weight gains
compared with that of placebo-treated animals. These animals
maintained body weight similar to normal non-tumour-bearing
mice despite the presence of tumour. It is noticeable that
tumour burdens in both curcumin- and placebo-treated animals
were not statistically different (P,0·67); therefore, the effect
of curcumin on body weight is independent of tumour burden.
The effect of curcumin c3 on body-weight gain was also inde-
pendent of food intake as there was no statistically significant
difference between the food intake of control and curcumin
c3-treated animals. Furthermore, the effects of curcumin c3
treatment on inhibiting and/or reversing cachexia in MAC16
tumour-bearing mice are also evident by weight gain in gasc-
trocnemic muscle (30–58%), and increased size of muscle
fibres (30–65%). It is interesting to note that the present
Table 1. Effect of curcumin c3 treatment on MAC-16 tumour-bearing mice
(Mean values with their standard errors)
Daily food intake (g)†
Tumour weight (g)
Body weight – tumour weight (g)
Gastrocnemius muscle weight (g)
a,b,cMean values within a row with unlike superscript letters were significantly different (P,0·05).
*Analysed by ANOVA for at least five animals per group.
†The daily food intake of the non-tumour-bearing animals was 2·47 (SE 0·09) g.
Fig. 3. Effect of curcumin c3 on muscle fibre size. (a) Transverse serial sec-
tions of quadricep muscles (10mm) were prepared. The sections were
stained using haematoxylin and eosin staining and pictures of rectus femoris
muscle section were recorded using a digital camera. (b) Surface area of
individual muscle bundles (ten mixed fibre types from four different viewing
sites at random in each group) was measured using ImagePro software
(Cybernetics, Silver Spring, MD, USA). Values are means for five animals
per group, with standard errors represented by vertical bars. The results
were analysed using ANOVA.a,b,cMean values at day 21 with unlike letters
were significantly different (P,0·001). Using Tukey’s multiple-comparison
test, significant differences were found between all groups (P,0·05). BW,
Attenuation of proteolysis by curcumin971
British Journal of Nutrition
results are not in agreement with previous studies on the effect
of curcumin on the cachectic MAC16 mouse model(34). How-
ever, there are technical differences in the present study com-
pared with previous studies(34). In previous studies, a drastic
loss in muscle mass or body weight was induced by the
tumours in a shorter period of time (20% over 5d)(34),
while we established a gradual loss in body weight by
implanting a smaller load of MAC16 cultured cells. This
approach resulted in a slow body-weight loss totalling
18–20% over 21d. Furthermore, we used a different source
Fig. 4. Effect of curcumin c3 on 20S chymotrypsin-like proteasome activity and muscle atrophy F-box (MAFbx)/atrogen-1 and muscle RING finger 1 (MURF-1)
expression in muscle. Gastrocnemius muscle homogenate was used to determine 20S chymotrysin-like activity and MAFbx/atrogen-1 and MURF-1 expression at
the end of the study (day 21 on curcumin treatment). (a) Chymotrysin-like 20S proteasome activity in the supernatant fraction of muscle homogenates was deter-
mined using a kit (20S Proteasome Activity Kit; Boston Biochem, Cambridge, MA, USA). (b) The solubilised protein extracts of muscle homogenates were used to
determine MAFbx/atrogen-1 and MURF-1 expression by Western blot analysis. Lanes 1, 2 and 7 represent MAC16–curcumin c3-treated mice (250mg/kg body
weight (BW)); lanes 3, 5 and 9 represent MAC16–curcumin c3-treated mice (100mg/kg BW); lanes 4, 6 and 8 represents MAC16-untreated mice. (c) Quantifi-
cation of protein expression (B, MAFbx/atrogen-1;
, MURF-1) was determined by densitometry analysis. Densities (arbitrary units) are shown underneath each
band whereas the numbers in parentheses refer to densities normalised to the loading control (glyceraldehyde 3-phosphate dehydrogenase; GADPH). Values are
the means for three experiments, with standard errors represented by vertical bars. The results were analysed using ANOVA.a,b,cMean values with unlike letters
were significantly different (P,0·05).
R. A. Siddiqui et al. 972
British Journal of Nutrition
of curcumin, i.e. curcumin c3 complex, which has been exten-
sively used in human trials(39–41). The composition of curcu-
min extracts in the previous study was not reported; it is
therefore not clear if there were compositional differences in
our curcumin c3 complex from that used in previous studies.
It is possible that purity and compositional differences may
have contributed to the biological activity of the curcumin
c3 effects. Moreover, we suspended curcumin in olive oil,
whereas other studies used curcumin dissolved in dimethyl
sulfoxide and then diluted (100 £ ) in PBS(34). It is possible
that olive oil may serve as a better vehicle for curcumin c3
complex as it is hydrophobic in nature and provides improved
bioavailability compared with an aqueous vehicle such as PBS
used in the previous study. Lastly, the animals in the present
study were on curcumin c3 treatment for 21d, whereas pre-
vious studies used a 4–5d treatment(34,37). Treatment over a
longer period of time with curcumin c3 might have overcome
the low bioavailability of curcumin in general. Several inves-
tigators have reported low plasma levels of curcumin and its
metabolites(38,42). Recent studies demonstrate that curcumin
and its metabolites are cleared from plasma in rats within
2h of oral treatment(42). During the present investigation,
the concentration of curcumin was not measured because it
was not possible to withdraw blood from these mice on a
daily basis during the 21d treatment. In our opinion, these fac-
tors, including the rate of body wasting, the unique patented
composition of curcumin c3 complex, its suspension in olive
oil and the duration of treatment might have facilitated the
efficacy of curcumin c3 complex.
The ubiquitin–proteasome pathway is the primary pathway
involved in protein catabolism and is felt to be the major
degradation pathway involved in various cachectic con-
ditions(17,47–50). The ubiquitin–proteasome pathway is stimu-
lated by TNF, IL-1, interferon g and other pro-inflammatory
mediators(51–54). Pro-inflammatory cytokines and proteol-
ysis-inducing factor activate the ubiquitin–proteasome path-
way through the transcription factor NF-kB. We tested 20S
chymotypsin-like activity of the ubiquitin–proteasome path-
way in muscle specimens of curcumin c3- or placebo-treated
animals. The present results indicate that chymotrysin-like
activity of the 20S proteasome was significantly suppressed
in mice treated with curcumin c3 compared with placebo-
treated mice. However, there was no difference on inhibition
of chymotrysin-like activity between low and high doses of
curcumin c3 treatment. During the present investigation, we
did not examine the chymotrysin-like activity of the 20S
proteasome in non-tumour-bearing muscle; it is therefore not
clear if the inhibition of chymotrypsin-like activity of the
20S proteasome reached a basal level on curcumin treatment.
Nevertheless, the present results are consistent with previous
findings where curcumin attenuated the proteolysis-inducing
factor-induced increase in the ‘chymotrypsin-like’ enzyme
activity of the 20S proteasome(34).
Genes encoding for ligases (i.e. atrogin-1/MAFbx and
MURF-1) within the ubiquitin–proteasome pathway are
instrumental in the development of muscle atrophy(17). For
example, the development of atrophy and muscle proteolysis
during sepsis is blocked by proteasome inhibitors(55). We
tested the expression of atrogin-1/MAFbx and MURF-1 in
muscles isolated from curcumin- and placebo-treated animals.
Expression of MURF-1 was inhibited in the muscle of animals
treated with high doses of curcumin c3, but there was no sig-
nificant effect on atrogin-1/MAFbx expression. It is not clear
from these results if curcumin inhibited proteolysis through
atrogin-1/MAFbx- and MURF-1-mediated pathways. It is
possible that other cellular pathways, including pathways for
protein synthesis, may be involved in mediating curcumin
effects on body-weight regulation. Several pathways have
been proposed for the effect of curcumin c3 in directly regu-
lating protein degradation and synthesis. For example,
expression of MAFbx/atrogin-1 and MURF-1 under stressing
conditions is regulated byunphosphorylatedforkheadboxtran-
scription factors class O (FoxO). Phosphorylation of FoxO is
regulated by phosphatidyl inositol 30-kinase (PI30K)-dependent
protein kinase B (AKT) activity, which inhibits protein degra-
dation by inhibiting MAFbx/atrogin-1 and MURF-1 expression
and diverts signals for protein synthesis(56–58). Atrogin-1/
MAFbx expression is also up-regulated via a p38 mitogen-acti-
vated protein kinase (MAPK)-dependent mechanism in C2C12
myotubes(59), while MURF-1 expression is stimulated through
an NF-kB-dependent mechanism(45,60,61). Curcumin has been
shown to prevent activation of NF-kB and prevent sepsis-
induced muscle protein degradation(62). Furthermore, curcumin
dation of inhibitor of NF-kB (IkBa)(63), which results in an
increase in the rate and extent of muscle regeneration after
trauma(36). In contrast to these studies, elevated activity of
to dietary curcumin treatment(64). The mdx mice have impaired
ties, which suggests the involvement of these enzymes in the
lack of curcumin effect on NF-kB activity(64). Curcumin
also has a p38-inhibiting property(65), which has been
shown to obstruct p38-mediated TNF-a up-regulation of atro-
gin-1/MAFbx in C2C12 myotubes(59). Similarly, the adminis-
tration of curcumin daily for four consecutive days before LPS
injection blunted LPS stimulation of atrogin-1/MAFbx mRNA
expression in mice muscle(35); however, these experiments
failed to show curcumin regulation of atrogin-1/MAFbx
expression through mediating AKT activity during LPS stimu-
lation. Based on these data, one could speculate that curcumin
regulates both protein degradation and synthesis pathways
possibly through regulating AKT, NF-kB and/or p38 MAPK
the molecular effects of curcumin on the regulation of these
In conclusion, treatment of cachectic animals bearing
MAC16 tumours with curcumin c3 resulted in the prevention
and reversal of cachexia. Curcumin c3 also attenuated 20S
proteasome activity but its effects on the inhibition of atro-
gin-1/MAFbx and MURF-1 expression are not clear. The
data presented in the present study imply that curcumin c3
may have an effective therapeutic or an adjuvant therapeutic
potential against cachexia.
The present study was supported by a grant from Abbott
USA). The present study does not include any product that
is sold by the sponsor, Abbott Nutrition.
Columbus, OH 43215,
Attenuation of proteolysis by curcumin 973
British Journal of Nutrition
R. A. S., T. D., P. M. and S. DeM. planned and supervised
the study. S. H., T. R. and K. A. H. performed in vivo and
in vitro experiments. The authors wish to thank Ms Diane
Bond for animal care; Ms Charlene Shaffer for secretarial
assistance and Dr Karen Spear and Heather Richardson for
editing the manuscript.
There is no potential conflict of interest by any of the
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