Inhibitory effect of curcumin on IMP dehydrogenase, the target for anticancer and antiviral chemotherapy agents

Article abstract of DOI:10.1271/bbb.90568

Inosine monophosphate dehydrogenase (IMPDH), a rate-limiting enzyme in the de novo synthesis of guanine nucleotides, is a therapeutic target for anticancer and antiviral agents. Among the 15 different polyphenols examined, curcumin was found to have an inhibitory effect on the IMPDH activity in both a competitive and uncompetitive manner and to suppress the cellular GTP level in HT-29 colon carcinoma cells.

Full text of DOI:10.1271/bbb.90568

Biosci. Biotechnol. Biochem., 74 (1), 185–187, 2010
Note
Inhibitory Effect of Curcumin on IMP Dehydrogenase, the Target
for Anticancer and Antiviral Chemotherapy Agents
y
Issei DAIRAKU, Yunkyung HAN, Noriyuki YANAKA, and Norihisa KATO
Graduate School of Biosphere Science, Hiroshima University, Higashi-Hiroshima 739-8528, Japan
Received August 3, 2009; Accepted October 2, 2009; Online Publication, January 7, 2010
[doi:10.1271/bbb.90568]
Inosine monophosphate dehydrogenase (IMPDH), a
rate-limiting enzyme in the de novo synthesis of guanine
nucleotides, is a therapeutic target for anticancer and
antiviral agents. Among the 15 different polyphenols
examined, curcumin was found to have an inhibitory
effect on the IMPDH activity in both a competitive and
uncompetitive manner and to suppress the cellular GTP
level in HT-29 colon carcinoma cells.
curcumin. After 72 h, the cell growth was evaluated by
an MTT assay.6) The cellular GTP levels were deter-
mined by an HPLC analysis according to the method
reported.7) All data are expressed as the mean ꢂ SE, and
all were analyzed by Scheffe’s multiple-range test
(Excel Statistics 2006 for Windows, Social Survey
Research Information Co., Tokyo, Japan). Results are
considered significant with a value of p < 0:05.
We investigated the inhibition of IMPDH by various
polyphenols. As shown in Fig. 1, among the polyphe-
nols examined, curcumin (68% inhibition), epigalloca-
techin gallate (47% inhibition), chlorogenic acid (30%
inhibition) and ellagic acid (27% inhibition) effectively
inhibited the activity of IMPDH at a concentration
of 100 mM. Resveratrol, genistein, eugenol, gingerol,
naringenin, naringin, caffeic acid, morin, kaempferol,
hesperidin and rosmarinic acid had no effect on the
activity of IMPDH (data not shown). The dose-
dependent inhibition by curcumin was observed at
concentrations of 0–200 mM with an IC50 value of
43.0 mM, at least 20 mM being necessary for significant
inhibition. The IC50 values for epigallocatechin gallate,
chlorogenic acid and ellagic acid were 112.5 mM,
>200 mM and >200 mM, respectively.
To obtain further information for the inhibition of
IMPDH by curcumin, the extent of inhibition as a
function of the IMP substrate concentration was studied
(Fig. 2). A Lineweaver-Burk plot indicated that the
Km value remained unchanged by different concentra-
tions of curcumin, while Vmax increased with increas-
ing curcumin concentration (0, 50, 75, and 100 mM).
The results were Km ¼ 50:9 mM and Vmax ¼ 0:063
mmoles/min/ml. The Ki value was determined to be
99.7 mM (data not shown). These plots suggest the
manner of inhibition to be both competitive and
uncompetitive. This is similar to such well-known
competitive and uncompetitive IMPDH inhibitors as
mycophenolate mofetil, mizoribine and ribavirin with
respect to IMP.8) To elucidate in detail the interaction
between IMPDH and curcumin, it will be necessary to
examine the X-ray crystal and NMR analyses of the
complex of IMPDH and curcumin.
Key words: IMP dehydrogenase; polyphenol; curcumin;
anticancer agent; antiviral agent
The enzyme, inosine monophosphate dehydrogenase
(IMPDH), catalyzes the nicotinamide adenine dinucleo-
tide (NADþ)-dependent oxidation of inosine mono-
phosphate (IMP) to xanthine monophosphate (XMP) in
de novo synthesis.1) This enzyme is a critical determi-
nant of the level of guanine nucleotides, including
guanosine triphosphate (GTP), that are essential for
many cellular functions.2) Inhibitors of IMPDH are
sought as potential pharmacological anticancer and
antiviral agents.3,4) The consumption of polyphenolic
compounds is believed to suppress the development of
cancer through mechanisms including reducing the
inflammatory process, and hyperproliferation.5) We
examined in the present study the influence of various
polyphenols on the activity of IMPDH.
IMP and NADþ were purchased from Sigma Aldrich
(St. Louis, MO, USA), and curcumin, resveratrol,
genistein, eugenol, gingerol, chlorogenic acid, epigallo-
catechin gallate, naringenin, naringin and caffeic acid
were purchased from Nacalai Tesque (Kyoto, Japan).
Morin, kaempferol, hesperidin and ellagic acid were
purchased from Wako Pure Chemicals (Osaka, Japan)
and rosmarinic acid was purchased from MP Biomed-
icals (Ohio, USA).
The cloning of mouse IMPDH type II and isolation of
recombinant mouse IMPDH were conducted according
to our previous report.1) The assay procedure for
IMPDH was described in the same report,1) in which
various polyphenols were added to the assay mixture by
dissolving in 10% dimethyl sulfoxide at 1 mM. Colon
carcinoma cell line HT-29 cells were maintained in
Dulbecco’s modified Eagle’s medium (Sigma Aldrich,
St. Louis, MO, USA) supplemented with 10% fetal calf
serum, 100 units/ml of penicillin, and 100 mg/ml of
streptomycin at 37 ꢀC in 5% CO2. A cell suspension
(3 ꢁ 103 cells/ml) was cultured for 48 h, and then the
medium was replaced with a culture medium containing
To examine the effect of curcumin on the GTP level
in colon cancer cells, HT-29 cells were treated with
curcumin at 50, 75, and 100 mM concentrations for 48 h
(Fig. 3). Curcumin significantly reduced the level of
GTP at the concentrations of 75 and 100 mM, implying a
suppressive effect of curcumin on the GTP pool.
IMPDH inhibition results in a depletion of the guanine
y
To whom correspondence should be addressed. Fax: +81-82-424-7916; E-mail: nkato@hiroshima-u.ac.jp

186
I. DAIRAKU et al.
nucleotide pool, with subsequent decreased DNA and
RNA synthesis.9) Increased IMPDH activity is correlated
with increased cellular proliferation and transforma-
tion.1) In our MTT assay, the relative values of cell
growth (%) at 0, 50, 75, and 100 mM of curcumin (n ¼ 5)
were 100 ꢂ 1, 31:6 ꢂ 0:3, 26:2 ꢂ 0:4, and 17:0 ꢂ 0:2,
respectively (curcumin effect at 50–100 mM, p < 0:05).
These results indicate that 50 mM curcumin had no effect
on cellular GTP, but significantly suppressed the cell
growth. Thus, the reduction in cell growth by higher
curcumin levels (75 and 100 mM) appears to be associ-
ated with the reduced GTP level. Curcumin, epigallo-
catechin gallate, chlorogenic acid and ellagic acid,
which were found to inhibit IMPDH activity in this
study, have been reported to possess anticancer activity
via cell proliferation inhibition10–13) and antiviral activ-
ity.14–17) We speculate that the anticancer and antiviral
mechanisms of these polyphenols might be, at least in
part, mediated by some inhibition of IMPDH. Further
study is necessary to test this possibility.
It has been reported for mice fed on a 0.2% curcumin-
containing diet that the steady-state levels of intact
curcumin were 111 mM in the small intestinal mucosa
and 508 mM in the colonic mucosa, although the serum
level was 5 nM.18) The dose of curcumin in humans
corresponding to a dietary 0.2% curcumin level used in
mice is 1.6 g/d, when calculated on the basis of a body
surface area of 1.8 m2 accompanying a body weight of
70 kg in humans.18,19) This is close to a putative
efficacious clinical dose of curcumin of 1.2–1.8 g/d in
humans.20) Thus, the curcumin level achieved in the
intestinal tract by an oral administration appears to be
enough to inhibit the IMPDH activity, but not enough to
affect the serum level.
*
*
*
*
*
*
Fig. 3. Effect of Curcumin on the Level of Cellular GTP in HT-29
Cells.
The effect of curcumin on the level of GTP in HT-29 cells was
determined by incubating cells treated with curcumin (50, 75, and
100 mM). Each value is the mean ꢂ SE, n ¼ 3. ꢃSignificantly
different from the control value (p < 0:05).
Fig. 1. Inhibitory Effect of Some Polyphenols on IMPDH Activity.
The concentration of polyphenols examined was 100 mM. The
enzyme activity in the absence of polyphenols was taken as 100%.
Each value is the mean ꢂ SE, n ¼ 4{6. ꢃSignificantly different from
the control value (p < 0:05).
100
70
M
M
50
0
M
M
1/[S]
M
-1 IMP
Fig. 2. Kinetic Analysis of the Inhibition of IMPDH by Curcumin.
The IMPDH activity was measured in both the absence and presence of curcumin by using the indicated concentrations of the substrate IMP.
The inhibitory mechanism was determined as the mixed inhibition by a Lineweaver-Burk plot. The amount of mouse IMPDH in the assay
mixture was 8 munits.

Inhibitory Effect of Curcumin on IMP Dehydrogenase
187
9) Floryk D and Thompson TC, Int. J. Cancer, 123, 2294–2302
(2008).
In summary, the results of this study provide the first
evidence for the inhibitory effect of curcumin on the
activity of IMPDH, the target for anticancer and
antiviral agents. This finding implies a novel function
for a polyphenol that is favorable to health.
10) Zhu BH, Zhan WH, Li ZR, Wang Z, He YL, Peng JS, Cai SR, Ma
JP, and Zhang CH, World J. Gastroenterol., 13, 1162–1169 (2007).
11) Losso JN, Bansode RR, Trappey AII, Bawadi HA, and Truax R,
J. Nutr. Biochem., 15, 672–678 (2004).
12) Thangapazham RL, Sharma A, and Maheshwari RK, AAPS J.,
8, 443–449 (2006).
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