Inhibition of glutathione reductase by plant polyphenols

Biochemical Pharmacology, Vol. 54, pp. 1047-1053, 0 1997 Elsevier Science Inc. All rights reserved.

ISSN 0006-2952/97/$17.00 + 0.00 PII SOOO6-2952(97)00315-8

1997.

ELSEVIER

Inhibition of Glutathione Reductase by Plant Polyphenols Kai Zhang, *f Er-Bin Yang,* Wen-Ying Tang,* Kim Ping Wong# and Peter Ma& DEPARTMENTS OF *EXPERIMENTAL SURGERYAND $SURGERY,SINGAPOREGENERALHOSPITAL,SINGAPORE169608; AND $DEPARTMENT OF BIOCHEMISTRY, NATIONALUNIVERSITY OF SINGAPORE, SINGAPORE 119260 ABSTRACT. The effects of forty-one plant polyphenols on the activity of glutathione reductase (GSH-RD) were studied. These polyphenols showed varying degrees of concentration-dependent inhibition on the enzyme, and tannic acid were with ICKYvalues that varied from approximately 40 FM to 1 mM. 4’.Hydroxychalcone among the more potent inhibitors, with ICKYvalues of 47.3 and 50.4 FM, respectively. Different classes of polyphenols varied in potency in the following order: chalcones > tannic acid > flavonoids > coumarins > catechins. Analysis of structure-activity relationships showed certain chemical structures to be important for the inhibition of GSH-RD: (a) C-5 and C-7 h yd roxylations in the A-ring, a carbonyl group at C-4, and the B-ring attached to C-2 in flavonoids; (b) C-2’ and C-4’ hydroxylations in chalcones; and (c) C-6 and C-7 hydroxylations in coumarins. The inhibition of GSH-RD by tannic acid and quercetin was time dependent and irreversible, whereas that by 4’-hydroxychalcone and esculin was reversible but not time dependent. Enhanced inhibition of GSH-RD by the four polyphenols 4’-hydroxychalcone, quercetin, butein, and acacetin was observed in the presence of NADPH. Kinetic studies showed that both tannic acid and 4’-hydroxychalcone exhibited non-competitive inhibition on GSH-RD towards glutathione disulfide. BIOCHEMPHARMACOL54;9: 1047-1053, 1997. 0 1997 Elsevier Science Inc. KEY WORDS. glutathione

GSH” and GSH-dependent detoxification of endobiotics many

anticancer

GSH

and overexpression

alkylating

confer

chemo-resistance

agents

[3, 41. Depletion

and inhibition the

drug-resistant

which

catalyzes

form, is important GSH

been

shown

[l,

21. High been

cells

against

by buthionine

by ethacrynic to these

in levels

of many anticancer

transport of GSH may contribute

cells to chlorambucil.

sensitize to the

actions on human colon adenocarcinoma bucil. The

inhibition

This study investigated

tumor

for GSH also has

MATERIALS Chemicals

AND

tissues

mechanisms

GSSG,

drugs [lo]. Some flavonoids [ll].

Interestingly,

found that flavonoids and other polyphenols

these plant

polyphenols

cells to chloram-

may also be involved.

the inhibitory effects of a variety of

on GSH-RD.

2) were obtained

concentrations

of GSH-RD

polyphenols

Flavonoids

and non-toxic

tumor

these effects alone were

not of a sufficient magnitude to account for their sensitizing

enzyme may be associated with tumor growth and resistance

human colon adenocarcinoma

However,

the high levels of is essential

in human

have been shown to inhibit GSH-RD

by human colon tumor cells

in our laboratory [ 13, 141, and this

to their ability to sensitize human

and in mouse tumor cell lines, and a high activity of the against anticancer

conjugates

of

alkylating

drugs [9]. GSH-RD

to be overexpressed

inhibition

to

[5, 61. GSH-RD,

in maintaining

polyphenols;

were also demonstrated

sulfoximine

of oxidized-GSH

in cells [7, 81. The latter

the

reported

acid could

drugs

chalcones;

including

have

agents

reduction

reduced

participate

xenobiotics

to tumor

reduced

conjugation

and

of GSH

cells the

enzymes

of GSTs

of GSTs

reductase; flavonoids;

we

could sensitize

cells to chlorambucil

(see Table 1) and other polyphenols

NADPH,

from Extrasynthese and GSH-RD

(Genay,

(see Table France).

(Type VII, from bovine

mucosa) were purchased from the Sigma Chem-

ical Co. (St. Louis, MO, U.S.A.).

at low

[12]. The inhibitory effects of

on rat liver GSTs

intestinal

METHODS

and on the

Determination GSH-RD

t Corresponding author: Dr. Kai Zhang, Department of Experimental Surgery, BLK 9, Level 2, Singapore General Hospital, Outram Road, Singapore 169608, Republic of Singapore. Tel. 65-321-3888; FAX 652223389; E-mail: [email protected] ” Abbreviations: GSH, glutathione; GSH-RD, glutathione reductase; GSTs, glutathione S-transferases; and GSSG, glutathione disulfide. Received 27 February 1997; accepted 30 May 1997.

Racker

of QSH-RD

Activity

activity was assayed according [15]. The

reaction

potassium phosphate

mixture

to the method of

consisted

buffer containing

of 50 mM

1 mM EDTA,

pH

7.4, with final concentrations of 1 mM GSSG and 0.2 mM NADPH. The reaction was started by the addition of NADPH. Consumption of NADPH was monitored at 37” for 3 min on a spectrophotometer

at 340

nm. Enzyme

1048

K. Zhang et al.

of NADPH

oxidized

per minute per milligram of protein at 37”, extinction coefficient of 6.22 mM_i cm-‘.

activity was expressed as nanomoles

using an

Kinetic

Studies

Kinetics

of the inhibition

concentrations lnhibition

Studies of the inhibition

of GSH-RD

by polyphenols

were

as a substrate.

The

were prepared

10 mM stock solutions of polyphenols in absolute

alcohol

or DMSO.

Then

aliquots

of

these solutions were added to the reaction mixture to yield a final concentration

ranging from 10 FM to 1 mM. In all

assays, the final concentration

of ethanol or DMSO did not

exceed

2%

(v/v), and this concentration

DMSO

did not affect GSH-RD

were calculated

activity by tannic

were studied using various

(0.05 to 1 mM) of GSSG

in the presence of

40 or 80 FM tannic acid and 4’-hydroxychalcone,

Studies

carried out at 37” and pH 7.4, using GSSG freshly

of GSH-RD

acid and 4’-hydroxychalcone

of ethanol

activity.

by linear regression

points in the range of 20 to 80%

The

or

ICKYvalues

of no less than five

inhibition.

Each point

represents the mean of at least two determinations.

respec-

tively.

RESULTS EfJects of PoZyphenots on CjSH-RD Forty-one GSH-RD

polyphenols

Activity

were tested

for their

effects

on

activity. Their potencies differed widely in inhib1c50 values calculated

from linear

regression for flavonoids and other polyphenols

are listed in

iting GSH-RD Tables

activity;

1 and 2, respectively.

inhibition

Concentration-dependent

was observed for most of the polyphenols

In the range of 20 to 80% inhibition,

tested.

the concentration-

response curves were found to be linear, with correlation coefficient

values between 0.92 and 0.99.

Time-Dependent Inhibition of CjSH-RD by Polyphenols In these experiments, 4’.hydroxychalcone,

the concentrations quercetin,

of tannic

stant at 30, 30, 60, and 120 p-M, respectively, GSH-RD

was incubated

periods:

10 min,

GSH-RD

with these inhibitors

30 min,

activity was determined

studies

GSH-RD (50

PM),

acacetin

as described above.

carried

out

by

preincubation

of

4’-hydroxychalcone

were determined

in the absence and compared

or presence

of

using Student’s

t-test 1161.

experiments CA, U.S.A.),

were carried

out

using standard

Medical Industries, Los

having cut-off of molecular

in the range of 12,000-14,000. protein/ml)

was inhibited

at 25” for 20 up to 79, 85, 80 p.M tannic

and 400 PM esculin, respectively

mixtures.

hydroxychalcone cantly

The

inhibition

and esculin

after dialysis for 24 hr (P

activities

of GSH-RD

could

by 4’-

be reversed

signifi-

O.OOOl),

but the

<

still could not be restored to the control

The inhibition

by tannic acid and quercetin

ible. Dialysis of the reaction

level.

was irrevers-

incubates for 24 hr was unable

to restore

the inhibited

GSH-RD

inhibition

was enhanced

significantly

activity.

In fact,

the

after 24 hr dialysis

Fig. 1).

of Inhibition

cellulose dialysis tubing (Spectrum Angeles,

with polyphenols

57, and 76% by 80 FM 4’-hydroxychalcone,

(P < 0.0001,

Reversibility Dialyzing

was incubated

min. The activity of GSH-RD

reaction

(100 FM), butein (100 FM), and (100 p.M) in the absence or presence of NADPH. of GSH-RD

GSH-RD

(Fig. 1). The inhibitors were then removed by dialyzing the

of GSH-RD

acid (50 FM),

of the Inhibition of CjSH-RD by PolyphenoE Reversibility

acid, 100 p,M quercetin

quercetin

The activities NADPH

were

with tannic

and then for various

1 hr, 2 hr, and 4 hr. Then

Effects of NADPH on the inhibition These

acid,

and esculin were held con-

The

GSH-RD

weight

(0.45

pg

was incubated in aliquots of dialyzing buffer (4

mL) containing

one of the following

compounds:

acid (80

FM),

4’-hydroxychalcone

(100

and esculin (400 p-M) at 25” for 20 min. (500 ~.LL) o f each incubation mixture were re-

(80

PM),

tannic

quercetin

PM),

Aliquots moved, and GSH-RD

activity

was measured. Additional

of the incubation

mixture

aliquots of the remaining

incu-

bation mixture (3.5 mL) were dialyzed against the dialyzing buffer which consisted of 50 mM potassium phosphate and 1 mM EDTA, pH 7.4, at 4” for 24 hr. GSH-RD activities were also determined after dialysis. The controls were without inhibitors and were treated in the same way as described for the test samples.

Time-Dependent Inhibition PIunt Polyphenoki Time-course

experiments

hydroxychalcone,

of GSH-RD

b

were also conducted

tannic acid, quercetin,

with 4’.

and esculin. In all

cases, GSH-RD activity fell rapidly over the first 10 min. At 10 min, tannic acid was the most potent inhibitor. However,

after

10 ruin there

were differences

in the

inhibitory behavior of the four compounds. For tannic acid and quercetin,

their inhibitory

effects increased

gradually

over 4 hr. At 4 hr, the inhibition of GSH-RD by 30 PM tannic acid was 97% as compared with 21% at 10 min. The of GSH-RD by quercetin time-dependent inhibition showed a pattern

similar to that of tannic

acid. Fifteen

percent and sixty-two percent inhibition were observed at 10 min and 4 hr, respectively. In contrast, the inhibition of GSH-RD by 4’-hydroxychalcone and esculin hardly changed over the period from 10 min to 4 hr (Fig. 2).

Inhibition

of GSH-RD

1049

by Plant Polyphenols

of GSH-RD by flavonoids*

TABLE I. Inhibition

Flavone

Flavonol

Flavanone

:wc q-p qp 0 Class

Name

0

Hydroxylation pattern

Acacetin Naringenin Quercitrin Fustin Quercetin Apigenin Isoquercitrin Diosmin

Flavone Flavanone Flavonol Flavanone Flavonol Flavone Flavonol Flavone

577 5,7,4’ 5,7,3’,4’ 3,7,3’,4’ 3,5,7,3’,4’ 5,7,4’ 5,7,3’,4’ 5,3’

Luteolin Myricetin Isorhamnetin Daidzein Genistein Morin Myricitrin Kaempferol Apiin Fisetin Galangin 7-Hydroxyflavanone Biochanin A Diosmetin Naringen

Flavone Flavonol Flavone Isoflavone Isoflavone Flavonol Flavonol Flavonol Flavone Flavone Flavone FIavanone Isoflavone Flavone Flavanone

3,5 3,5,7,3’,4’,5’ 3,5,7,4’ 7,4’ 5,7,4’ 3,5,7,2’,4’ 3,5,7,4’,5’ 5,7,4’ 5,4’ 3,7,3’,4’

4’-HydroxyfIavanone Flavanone (+)CarechinS (-)Epicatechin$

Flavanone Flavanone Flavan-3-01 Flavan-3-01

4’

Substitution

C-3

63.5 65.6 69.4 72.2 73.2 75.2 84.5 89.4

= -rhamnose

C-3 = -glucose C-7 = -rutinose C-4’ = -OCH, C-4‘ and C-7 = -OCH,

94.0 96.8 104.5 113.5 116.3 118.7 124.7 126.5 128.3 129.7 180.4 268.2 276.2 279.7 313.8

C-3’ = -OCH, B-ring at C-3 B-ring at C-3 C-3

= rhamnose

C-4

= -0

-

glucose

-

apit

3157 B-ring at C-3 C-4’ = -OCH, C-4 = -OCH, C-5 = rhamnose

:7 517,3’ 7,4’

-

glut 580.7 869.1 >lOOO >lOOO

3,5,7,3’,4’ 3,5,7,3’,4’

* The flavanoid concentrations used were m the range of 10 FM to 1 mM. GSH-RD activity was measured according to the method of Racker [15]. The I+, values were calculated by Linear regression of no less than five points in the range of 20.-80% inhibition. t -glu, -glucose; -api, -apiose. $ Flavonolds wthout a carbonyl group at C-4 and a double bond between C-2 and C-3.

Effects

on the Inhibition of

of NADPH

by PolyphemJzs GSH-RD

presence except cofactor cantly

or absence

with

0.01)

five

of NADPH.

polyphenols

As shown

acid, preincubation

required for GSH-RD (P <

DISCUSSION An analysis of the structure-activity

was incubated for tannic

GSH-RD

activity)

the inhibition

in the

in Table

with NADPH enhanced

that various classes of polyphenols

3,

order of potency

(a

cones

signifi-

by all the compounds

tested.

echins.

>

tannic

for the

inhibition

acid > flavonoids

A similar potency

inhibition

relationship exhibited

of GSTs

study, for chalcones,

indicated

the following

of GSH-RD: > coumarins

chal> cat-

order also was observed for the

by polyphenols

[13]. In the present

the hydroxylation

substitution

at the

C-4’ position was obviously associated with their inhibitory Inhibitory Kinetic 4’-Hydroxychalcone kinetic experiments.

potencies on GSH-RD

Studies of GSH-RD

(4’-hydroxychalcone,

etin, and phloredzin with hydroxylation

and tannic acid were selected for the From the Lineweaver-Burk plot anal-

potent inhibitors However,

than chalcone

butein, phlorat C-4’

are more

and 2’-hydroxychalcone).

in our previous report, for the inhibition

of rat

ysis, both tannic acid and 4’-hydroxychalcone showed non-competitive inhibition towards GSSG (V,,, de-

liver GSTs by chalcones, hydroxylation substitutions at C-2 and C-3 were more important [13]. Generally, as in the

creased, while K,

inhibition

remained unchanged)

(Fig. 3).

of other

enzymes

by plant

polyphenols

[13,

1050

K. Zhang et al.

TABLE 2. Inhibition of GSH-RD by other polyphenols

Compounds

Structure

rcso (ILM)

Tannic acid

50.4

COOH Gallic acid

818.6 HO

w

Coumarin

Daphnetin Esculin 2-Hydroxycoumarin

‘2

0

>lOOO

3

c-2

C-6

c-7

C-8

H H OH

H OH H

OH OH H

OH H H

234.5 288.2 >lOOO 97.0

Chalcone

4’-Hydroxychalcone Phloredzin Butein Phloretin 2’.Hydroxychalcone 2-Hydroxychalcone Marein

c-2

c-3

c-4

C-2’

C-3’

C-4’

C-6’

H H H H H OH H

H H OH H H H OH

H OH OH OH H H OH

H o-glu H OH OH H OH

H H H H H H OH

OH OH OH OH H H OH

H OH OH OH H H H

Thep&phenol

concentrations used wae in the range of 10 WM to 1 mM. The GSH-RD activity calculated by linear regression of no less than five points in the range of 20-80% inhibition.

17-191, hydroxylation

of the polyphenols is also an impor-

tant structural feature for the inhibition

of GSH-RD.

For

instance, tannic acid with polyhydroxylations is a potent inhibitor. The inhibitory effects of naringenin (5,7,4’trihydroxyflavanone), 7-hydroxyflavanone, and flavanone

was measured

comparing

the

according

to the method

potencies

of Racker

of flavanoids

47.3 68.1 70.8 71.5 82.0 123.2 176.1 [15]. The

~~~~ values

were

that

belong

to

different classes but possess identical hydroxylation patterns, the following order of potency was observed: flavanone

(naringenin)

>

flavone

(apigenin)

>

flavonol

are noted to be in a decreasing order of potency. In addition, when compared with coumarin, the inhibition by daphnetin and esculin (both of which are dihydroxycou-

(kaempferol). Interestingly, when the B-ring of the flavonoid molecule is attached to the C-3 position (i.e. isoflavone class), their inhibitory potencies decreased (e.g. daidzein, genistein, and biochanin A). Coumarins (without

marins)

B-ring) also exhibited

was enhanced

significantly

(Tables

1 and 2). By

reduced inhibitory

potency.

In sum-

Inhibition

of GSH-RD

1051

by Plant Polyphenols

TABLE 3. Effects by polyphenols

of NADPH

on the inhibition

of GSH-RD

Remaining activity (% of control)

+ Polyphenols

4’-Hydroxychalcone, 50 PM

51.2 11.7 24.7 18.7 52.8

Tannic acid, 50 p,M Quercetin, 100 pM Butein, 100 pM Acacetin, 100 pM Experiments

C

TA

Q

E

presence

were

tann,c

FIG. I. Effects of dialysis on the inhibition of GSH-RD by plant polyphenols. Experiments were carried out as described in “Materials and Methods.” GSH-RD activities after dialysis were significantly different from those before dialysis (*P < 0.0001). Control activities before and after dialysis were 149.7 and 141.9 nmol/min/mg protein, respectively. Values are means * SD of triplicate experiments. Abbreviations: C, 4’-hydroxychalcone; TA, tannic acid; Q, quercetin; and E, es&in. and C-7 hydroxylations

mary, C-5

group at C-4, C-2’

C-7

and the B-ring

and C-4’

hydroxylations

hydroxylations

features

for

in the A-ring,

the

are

gallic acid are poor inhibitors,

chemical

tures that are required for inhibitory GSH-RD

experiments

and

chemical

Catechins

and

possibly due to the absence

of some of the above-mentioned Dialysis

important

of GSH-RD.

inhibition

and C-6

in chalcones;

in coumarin

a carbonyl

to C-2 in flavanoids;

attached

showed

by 4’-hydroxychalcone

ible, but the activity of GSH-RD

structural

fea-

potency on GSH-RD.

that

the

inhibition

and esculin

of

was revers-

after dialysis still did not

return to the control level. This finding indicates that although the inhibition by these two compounds is revers-

perfwmed

or absence

was measured aad,

NADPH

as described

were

NADPH

were

ible,

m “Materials

and

SD vf three

151.1

recovery These

of the

by tannic

results

time-course

mcuhate

presence

with

of 100 PM (1’

and

respectively.

inhibitors

i

0.01,

abbence

of

V.~lues

are

to the

However,

acid and quercetin

are in accord

with

studies of the inhibition

ibility of inhibition

acrwity

enzyme

more drastic than dialysis for total

of the enzyme activity.

GSH-RD

for the

presence

prorem,

in the

GSH-RD

NADPH

the

8.80 1.12 2.99 3.24 3.45

polyphenol\

m the

wirhwr m

2 r -t 5 2

expermww.

attachment

requires a treatment

those

wth

Except

mcuhatr.\

actwities

76.9 13.8 43.7 32.6 67.0

at 25”. Then

Method>.”

other

nmol/mm/mg

mdeprndent

1.86 3.51 1.83 2.03 2.81

of GSH-RI>

from

GSH-RD

t + t -+ -+

for 60 mm and

of the

dlffeerent

Control 130.2

the

incubation NADPH

acnwt~es

significantly

t-test).

t

by

of 100 PM

GSH-Rl)

Student’s

meam

NADPH

NADPH

by tannic

inhibition

of

was irreversible.

those

obtained

of GSH-RD.

acid and quercetin

may be

explained

by their time-dependent

inhibition

at longer exposure time to the two compounds),

while the inhibition was reversible

inhibition

from

Irrevers-

by 4’-hydroxychalcone

but not

different mechanisms

time

dependent.

for the inhibition

(enhanced and esculin

This

suggested

of GSH-RD

by the

different classes of polyphenols. The inhibition

of GSH-RD

by the polyphenols

for tannic acid) was enhanced This suggested that inhibition pounds is dependent the reaction, NADPH, GSSG

of GSH-RD

the first step is reduction

of the GSH-RD

by

and then the enzyme catalyzes the reduction

tannic acid was not enhanced of NADPH.

significantly

As discussed above, inhibition

acid increased

inhibition

by these com-

on the redox state of the enzyme. In

to form GSH. However, inhibition

tannic

(except

in the presence of NADPH.

in the presence of GSH-RD

quickly with incubation

of GSH-RD

of

of the enzyme by by

time. The

by tannic acid after a l#hr incuba-

tion almost reached maximum, even without preincubation with NADPH NADPH

B

I

0 0

20

40

60

80

100 120 140 160 180 200 220 240 Time (min)

FIG. 2. Time-dependent inhibition of GSH-RD by plant polyphenols. GSH-RD was incubated with plant polyphenols for the indicated periods. GSH-RD activity was determined by the method described in “Materials and Methods.” GSH-RD activities of controls at 10 min, 30 min, 1 hr, 2 hr, and 4 hr were: 149.1, 147.6, 133.8, 128.2, and 119.4 nmol/min/mg protein, respectively. Data points are means of three independent experiments.

(Table

3). Therefore,

could not have a noticeable

tion of GSH-RD Involvement

preincubation

with

effect on the inhibi-

by tannic acid. of reactive

nism of inhibition

oxygen species in the mecha-

of GSH-RD

by dephinidin

chloride and

myricetin flavonoids has been described [l I]. Superoxide anion radicals might react with certain flavonoids (e.g. myricetin) inhibited

to form reactive GSH-RD.

intermediates,

However,

by morin and quercetin

which, in turn,

the inhibition

remained

of GSH-RD

the same under both

hypoxic and aerobic conditions [ll]. This suggested that the proposed oxygen-dependent mechanisms may work only for some individual flavonoids. Polyphenols have been

1052

K. Zhang et al.

0.16-

40 pM Tannic acid

l

FIG. 3. Inhibitory kinetics of GSH-RD by 4’hydroxychalcone and tannic acid. Experiments were carried out by various concentration of GSSG from 0.05 to 1 mM as described in Materials and Methods. Both tannic acid (top panel) and 4’hydroxychalcone (bottom panel) showed non-competitive inhibition towards GSSG (V_ decreased; K,,, remained unchanged). Data points are mean values of three separate experiments.

-6

-4

-2

0

2

4

6

8

IO

1 /[GSSG]

12

14

16

18

20

(mM)

0.07-

0.06 -

-8

-6

-4

-2

0

2

4

6

8

10

l/[GSSG]

shown to complex

with proteins

through

oxygen-independent of GSH-RD

mechanism involved in the inhibition

tion of drug-resistant

were developed

tumor cells. Verapamil,

its analogue,

PCS

conjugation

833,

could

for sensitizacyclosporin

reverse

cellular

14

agents.

and medicinal

properties

The

biological,

[24]. Quercetin

may be due to an increase [26], inhibition of protein

effective

zation of human tumor cells to anticancer

tance-associated

protein

mediated

(MRP)

by multidrug resis-

[23]. Our previous studies

pharmacological, have been have effects

properties of these of cellular cyclic kinase [27], and

of DNA, RNA, and protein synthesis [28]. These

effects of plant polyphenols

MDR

cells to these che-

growth-inhibitory

multidrug resistance (MDR) by blocking P-glycoproteinlinked efflux of drugs [21, 221. However, they were not in reversing

20

and tangeretin

on tumor cells [25]. The antineoplastic

inhibition

18

of these polyphenols

been shown to have preferential polyphenols AMP level

16

(mM)

and sensitize drug-resistant

motherapeutic

studied extensively

is a major problem in cancer chemother-

apy. Many chemosensitizers and

and

by some polyphenols.

Drug resistance

A,

hydrogen

[20]. This may be an

covalent bonds, causing precipitation

12

may also contribute

products found in many traditional

to sensiti-

drugs. As natural

herbal medicines

[29]

have shown that polyphenols could sensitize human colon adenocarcinoma cells by inhibiting GSTs and transport of drug-glutathione conjugates [12-141. In this in vitro study,

and in the human diet [30], these polyphenols should be more

polyphenols also inhibited glutathione reductase at micromolar concentrations. This inhibition of GSH-RD by plant polyphenols and a resulting low level of reduced GSH may decrease the detoxification of anticancer drugs by GSH

This work was supported by a grant from the National Medical Research Council of Singapme. The authors wish to thank Robert Ng, In-Ching Song, and Irene Kee of the Department of Experimental Surgery fur their technical assistance.

acceptable as chemosensitizers for cancer chemotherapy.

Inhibition

of GSH-RD

by Plant Polyphenols

References 1. Mannervik B and Danielson UH, Glutathione S-transferase: Structure and catalytic activity. CRC Crit Rev Biochem 23: 283-337, 1988. enzymes in anticancer drug 2. Tew KD, Glutathione-associated resistance. Cancer Res 54: 4313-4320, 1994. 3. Schecter RL, Alaoui-Jalami MA and Batist G, Glutathione S-transferases in chemotherapy resistance and carcinogenesis. Biochem CeEl Biol 70: 349-353, 1992. 4. Schneider E, Yamazaki H, Sinha BK and Cowan KH, Buthionine sulphoximine-mediated sensitisation of etoposide-resistant human breast cancer MCF7 cells overexpressing the multidrug resistance-associated protein involves increased drug accumulation. Br J Cancer 71: 738-743, 1995. 5. Godwin AK, Meister A, O’Dwyer PJ, Huang CS, Hamilton TC and Anderson ME, High resistance to cisplatin in human ovarian cancer cell lines is associated with marked increase of glutathione synthesis. Proc Nutl Acad Sci USA 89: 30703074, 1992. of glutathione 6. Chen G and Waxman DJ, Identification S-transferase as a determinant of 4-hydroperoxycyclophosphamide resistance in human breast cancer cells. Biochem Pharmacol49: 1691-1701, 1995. 7. Meister A and Anderson ME, Glutathione. Annu Rev Biochem 52: 711-760, 1983. 8. Commandeur JNM, Stijntjes GJ and Vermeulen PE, Enzymes and transport systems involved in the formation and disposition of glutathione S-conjugates. Role in bioactivation and detoxication mechanisms of xenobiotics. Pharmacol Reu 47: 271-330, 1995. 9. Colvin OM, Friedman HS, Gamcsik Ml’, Fenselau C and Hilton J, Role of glutathione in cellular resistance to alkylating agents. Adv Enzyme Regul 33: 19-26, 1993. 10. Muller JG, Bucheler US and Krauth-Siegel RL, Glutathione reductase in human and murine lung tumors: High levels of mRNA and enzymatic activity. Cell Mel Biol (Noisy&-grand) 39: 389-396, 1993. 11. Elliott AJ, Scheiber SA, Thomas C and Pardini RS, lnhibition of glutathione reductase by flavonoids: A structureactivity study. Biochem Pharmacol44: 1603-1608, 1992. 12. Zhang K and Wong KP, Glutathione conjugation of chlorambucil: Measurement and modulation by plant polyphenols. Biochem 1, 325: 417-422, 1997. 13. Zhang K and Das NP, Inhibitory effects of plant polyphenols on rat liver glutathione S-transferases. Biochem Pharmacol47: 2063-2068, 1994. 14. Zhang K and Wong KP, Inhibition of the efflux of glutathione conjugate by plant polyphenols. Biochem Phurmacol52: 163 l1638, 1996. 15. Racker E, Glutathione reductase (liver and yeast). In: Methods in Enzymology (Eds. Colowick SP and Kaplan NO), Vol. II, pp. 722-725. Academic Press, New York, 1955. 16. Snedecor GW and Cochran WC, The comparison of two samples. Statistical Metho& pp. 83-106. Iowa State University Press, Ames, IA, 1989.

1053

17. Merlos M, Sanchez RM, Camarasa T and Adzet T, Flavonoids as inhibitors of rat liver cytosolic glutathione S-transferases. Experientia 47: 616-619, 1991. 18. Kuppusamy UR and Das NP, Effects of flavonoids on several venom hyaluronidases. Experientia 47: 1196-1200, 1991. 19 Kuppusamy UR and Das NP, Effects of flavonoids on cyclic AMP phosphodiesterase and lipid mobilization in rat adipo, cytes. Biochem Pharmacol44: 1307-1315, 1992. 20 Haslam E and Lilley TH, Interactions of natural phenols with macromolecules. In: Plant Polyohenols in Biology and Medicine: Biochemical, Pharmacological, and Structure-Activity Relationships (Eds. Cody V, Middleton E, Harbome JR and Beretz A), pp. 53-65. Alan R. Liss, New York, 1986. 21 Ford JM and Hait WN, Pharmacology of drugs that alter multidrug resistance in cancer. Pharmacol Reu 42: 155-199, 1990. 22 Keller RP, Altermatt HJ, Donatsch P, Zihlmann P, Laissue JA and Hiestand PC, Pharmacological interactions between the resistance-modifying cyclosporin SDZ PSC 833 and etoposide (VP 16-123) enhance in viva cytostatic activity and toxicity. Int J Cancer 51: 433-438, 1992. 23 Barrand MA, Rhodes T, Center MS and Twentyman PR, Chemosensitization and drug accumulation effects of cyclosporin A, PSC-833 and verapamil in human MDR large cell lung cancer cells expressing a 190k membrane protein distinct from P-glycoprotein. Eur J Cancer 29A: 408-415, 1993. E and Kandaswami C, The impact of plant 24. Middleton flavonoids on mammalian biology: Implications for immunity, inflammation and cancer. In: Flawonoids: Aduunces in Research Since 1986 (Ed. Harborne JB), pp. 619-652. Chapman & Hall, London, 1994. 25 Kandaswami C, Perkins E, Drzewiecki G, Soloniuk DS and Middleton E, Differential inhibition of proliferation of human squamous cell carcinoma, gliosarcoma and embryonic fibroblast-like lung cells in culture by plant flavonoids. Anticancer Drugs 3: 525-530, 1992. 26. Graziani Y, Winikoff J and Chayoth R, Regulation of cyclic AMP level and lactic acid production in Ehrlich ascites tumor cells. Biochim Biophys Acta 497: 499-505, 1977. 27. Cunningham BDM, Threadgill MD and Hickman HA, Synthesis and evaluation of substituted flavones as inhibitors of tyrosine protein kinase. Br J Cancer 56: 207-213, 1987. 28. Graziani Y and Chayoth R, Regulation of cyclic AMP level and synthesis of DNA, RNA and protein by quercetin in Ehrlich ascites tumor cells. Biochem Phurmacol 28: 397-403, 1979. 29. Haslam E, Lelly TH, Ya C, Martin R and Magnolato D, Traditional herbal medicine. The role of plant polyphenols. Planta Med 55: 1-8, 1989. 30. Kuhnau J, The flavonoids: A class of semi-essential food components: The role in human nutrition. World Rep, Nutr Diet 24: 117-121, 1976.