enhanced chemopreventive effect by combining quercetin …...enhanced chemopreventive effect by...

Enhanced Chemopreventive Effect

by Combining Quercetin and Green

tea in Prostate Cancer

Piwen Wang, MD, PhD

Assistant Professor, Division of Cancer Research and Training

Charles R. Drew University of Medicine and Science

(Cancer Facts & Figures 2015)

Age-Standardized Prostate

Cancer Incidence and Mortality

Rates by World Area. Source:

GLOBOCAN 2008.

(Jemal A, et al. CA CANCER J CLIN 2011;61:69–90)

Prostate cancer

Prostate cancer: Science and clinical practice. Edited by: J Mydlo & C Godec. 2013)

Tea

From the plant Camellia sinensis

Green tea (GT):

- Not fermented

- Polyphenol monomer (EC, EGC,ECG, EGCG)

Oolong tea:

- Partially fermented

Black tea:

- Fully fermented

- Dimers and polymers

(theaflavins, thearubigins)

(Alipoor, et al. Asian J Clin Nutr. 2012, 4 (1): 1-15

GT and GT polyphenols

Anti-carcinogenesis

- Skin, lung, oral cavity, esophagus, stomach,

intestine, colon, liver, pancreas, bladder,

mammary gland, prostate

Diabetes and obesity

Cardiovascular diseases

Neurodegenerative diseases

Osteoporosis

(Yang CS, et al. Nature Reviews Cancer 2009, 9, 429-439)

Possible targets for the cancer preventive activity of (-)-epigallocatechin-3-gallate (EGCG).

Pooled odds ratio (OR) of GT consumption (highest

versus non/lowest) and risk of prostate cancer

(Zheng J. et al. 2011 Nutr and Cancer 63(5):663-672)

Intervention studies: GT and prostate cancer

Tea, Country Effect Reference

GT extract,

Italy,

600 mg

Reduced progression from

HG-PIN to prostate cancer:

Control 30%

GTE 3%

Bettuzzi S et al.

2006

GT extract, USA,

800 mg EGCG

(single arm)

Decreased serum PSA, HGF,

VEGF in prostate cancer

patients

McLarty J et al.

2009

GT extract, USA,

800 mg EGCG

(placebo

controlled)

A decreasing trend of PSA,

in prostate cancer patients

Nguyen MM et al.

2012

Screening

Randomize of eligible subjects

3-8 weeks intervention

Placebo (N=33) GT (N=34)

Urine

polyphenol

metabolites

8-OHdG

Serum

polyphenol

metabolites

ex vivo

bioassay

PSA

Prostate

polyphenol

metabolites

Prostate

proliferation

apoptosis

ROS

Bax/Bcl-2

NFkB

BT (N=26)

A phase II clinical trial of tea in prostate cancer

(Henning. et al. 2015 Prostate 75:550-559)

Limitations of GT in chemoprevention

EGCG 4"-MeEGCG ECG

Co

nc

en

tra

tio

n (

pm

ol/g

tis

su

e)

0

5

10

15

20

25

30

35

0h 24h 48h 72h

Ce

ll v

iab

ilit

y (

% o

f c

on

tro

l)

0

20

40

60

80

100

120

NT

4"-MeEGCG 40uM

EGCG 40uM

4"-MeEGCG 80uM

EGCG 80uM

a

ab

ab

abcd

ab

ab

abcd

abc

Low bioavailability of GT polyphenols

Extensively methylation in vivo

(Wang P, et al. 2010 Cancer Prev Res 3(8):985-93)

Catechol-O-methyltransferase (COMT)

CH3

EGCG

Quercetin (Q)

A flavonoid commonly found in fruits and vegetables - red

onions, apples, broccoli, berries, and tea

A natural inhibitor of catechol-O-methyltransferase (COMT) and

multidrug resistance-related proteins (MRPs)

Quercetin in combination with EGCG in vitro

– cellular absorption

EGCG 80uMQuer 5uM

Quer 10uM

EGCG + Quer 5uM

EGCG + Quer 10uM

Co

ncen

trati

on

(p

mo

l/m

g c

yto

so

lic p

rote

in)

0

100

200

300

400

500

600

700

800

900 EGCG

4"-MeEGCG

Quercetin

Isorhamnetin

a

a b

b

b

a

c

c

c

c

d

d

dd

LNCaP

EGCG 80uMQuer 10uM

Quer 20uM

EGCG + Quer 10uM

EGCG + Quer 20uM

Co

nc

en

tra

tio

n (

pm

ol/m

g c

yto

so

lic

pro

tein

)

0

100

200

300

400

500

600

700

800

EGCG

4"-MeEGCG

Quercetin

Isorhamnetin

a

a

a

b b

b

c

c

c

c

d

d

d

d

PC-3

The groups with the same superscript letter represent significant difference between groups (P<0.05)

(Wang P, et al. 2012 Nutr Cancer 64(4): 580-587)


– Antiproliferative effect

0 24 48

Ce

ll v

iab

ilit

y (

% o

f c

on

tro

l)

0

10

20

30

40

50

60

70

80

90

100

110

NT

EGCG 40uM

Quer 5uM

Quer 10uM

EGCG 40um + Quer 5uM


a

abcd

a

a

abc

abcd

aabcd

LNCaP

Time (Hours)


0 24 48

Cell v

iab

ilit

y (

% o

f co

ntr

ol)

0

10

20

30

40

50

60

70

80

90

100

110

NT

EGCG 40uM

Quer 10uM

Quer 20uM



abc

abc

ab

ab

abcd

abcd

PC-3

Time (Hours)

Compared to – a, control (NT); b, EGCG; c, Q 5umol/L; d, Q 10umol/L, p<0.05


– COMT inhibition

Time (Hours)

2 24 48

Van

illic a

cid

fo

rmed

(n

mo

l/h

/mg

pro

tein

)

0

10

20

30

40

50

60

70

80

90

NT

EGCG 40uM

Quer 10uM

Quer 20uM

EGCG 40uM + Quer 10uM


aa

b

c

d

e

a a

b

c

d

e

ac

b

d

e

f

NT EGCG

40 μM

Quer

10 μM

EGCG 40 μM+

Quer 10 μM

48h

COMT

β-actin 24h

COMT

β-actin

Time (Hours)

24 48

CO

MT

co

ncen

trati

on

(re

lati

ve t

o N

T)

0.0

0.2

0.4

0.6

0.8

1.0

1.2

NT

EGCG 40uM

Quer 10uM


a a

abc

a

abc


0h 24h 48h

Cell v

iab

ilit

y (

% o

f co

ntr

ol)

0

10

20

30

40

50

60

70

80

90

100

110

NT

EGCG 40uM

Quer 10uM

Quer 20uM



a

a

abcd

a

abc

abcd

abcd

abcd

A549 (lung cancer)

0h 24h 48h

Cell v

iab

ilit

y (

% o

f co

ntr

ol)

0

10

20

30

40

50

60

70

80

90

100

110

NT

EGCG 40uM

Quer 10uM

Quer 20uM



a

abc

abcd

aaa

abcd

abcd

786-O (kidney cancer)0h 24h 48h

Ce

ll v

iab

ilit

y (

% o

f c

on

tro

l)

0

10

20

30

40

50

60

70

80

90

100

110

NT

EGCG 40uM

Quer 10uM

Quer 20uM



abcd

abcda

aa

a

abc

abcd

Hep G2 (liver cancer)

Increased anti-proliferative effect by EGCG + Q in lung, liver

and kidney cancer cells

(Wang P, et al. 2012 Food Funct.3(6):635-42)

Xenograft SCID mouse model

0.5 million LAPC4 cells

When tumor was palpable - start of intervention

Weeks 0 1 2 3 4 5 6 7

Quercetin in combination with GT in SCID mice

GT (Celestial Seasonings, Boulder,

CO): 1 tea bag brewed in 240 ml

boiling water for 5min; 0.07% GT

polyphenols

Q (Sigma-Aldrich): 0.4% in diet EGC EGCG EC ECG

Co

ncen

trati

on

(m

g/L

wate

r)

0

100

200

300

400

500

AIN 93G+Quercetin

Xenograft tumor growth

Compared to – a, control group; b, Q group; c, GT group, p<0.05

Time (Weeks)

0 1 2 3 4 5 6 7

Tu

mo

r vo

lum

e (

cm

3)

0.0

0.2

0.4

0.6

0.8

1.0

1.2

Control

GT

Q

GT + Q

abc

a

a

a

a

abc

a

Tum

or

inocula

tion

Tre

atm

ent

sta

rted (21%)

(16%)

(45%)

(Wang P, et al. 2014 J Nutr Biochem 25(1):73-80)

Food and water consumption and body weight

Food WaterDa

ily c

on

su

mp

tio

n o

f fo

od

(g)

an

d w

ate

r (m

l) p

er

mo

use

0.0

2.0

4.0

6.0

8.0

Control

GT

Q

GT+Q

Weeks

0 1 2 3 4 5 6 7

mo

us

e w

eig

ht

(g)

16

18

20

22

24

26

Control

GT

Q

GT+Q

GT Q GT+Q

Concentr

ation (

pm

ol/g tis

sue)

0

10

20

30

40

50

60

70

80200

300

400

500

600

700EGCG

4"-MeEGCG

EGC

ECG

EC

quercetin

isorhamnetin

*

*

*

*

*


GT+Q increased the bioavailability and decreased

methylation of GT polyphenols in tumor tissues

GT Q GT+Q

Co

nc

en

tra

tio

n (

pm

ol/g

tis

su

e)

0

50

100

150

200

300

350

400

EGCG

4"-MeEGCG

EGC

ECG

EC

quercetin

isorhamnetin

Lung

*

**

*

*

*

GT Q GT+Q

Conce

ntr

atio

n (

pm

ol/g

tis

sue)

0

50

100

150

200

250

300

400

450 EGCG

4"-MeEGCG

EGC

ECG

EC

quercetin

isorhamnetin

Kidney

*

**

*

*

*

GT Q GT+Q

Co

nce

ntr

atio

n (

pm

ol/g

tis

sue)

0

100

200

300

400

500900

950

1000

1050

1100

EGCG

4"-MeEGCG

EGC

ECG

EC

quercetin

isorhamnetin

Liver

* *

*

* compared to individual treatment, p<0.05

(Wang P, et al. 2012 Food & Function 3: 635-642 )

GT+Q increased the bioavailability and decreased

methylation of GTPs in other tissues

COMT DNMT AR PSA VEGF

Pro

tein

conce

ntr

atio

n (

rela

tive t

o c

ontr

ol)

0.0

0.2

0.4

0.6

0.8

1.0

1.2

1.4

Control

GT

Q

GT+Q

* *

** *

**

***

*

*

**

Modulation of protein marker expression

GT Control GT+Q Quercetin

COMT

GAPDH

DNMT

AR

PSA VEGF


Inhibition of proliferation marker-Ki67


GT+Q in combination with drugs for

chemotherapy of advanced prostate cancer

GT+Q+Docetaxel: Enhanced inhibition of PC-3 tumor growth

Data are presented as mean±SE. Different letters at each time point indicate statistically

significant difference between groups, p<0.05 (manuscript submitted)

Weeks

0 1 2 3 4 5 6 7 8 9 10 11

Tum

or

volu

me (

cm

3)

0.0

0.2

0.4

0.6

0.8

1.0

1.2

1.4

1.6

Control

GT+0.4%Q

Doc 5

GT+0.4%Q+Doc 5

Doc 10

Tu

mo

r in

ocu

latio

n

Tre

atm

en

t sta

rte

da

ab

bcc

c

a

ab

b

b

b

a

a

ab

b

b

a

b

b

a

b

b

La

st

Do

c in

jectio

n

(100%)

(38%)

(34%)

Summary and Conclusion

Quercetin in combination with GT significantly enhances the

anti-carcinogenic effect in vitro and in vivo

The combined effect was associated with an increased

bioavailability and decreased methylation of GT polyphenols

These results warrant future studies to confirm the synergistic

effect of the combination of GT and quercetin in humans

In addition, this combination may improve clinical application

in treatment of advanced prostate cancer by sensitizing tumor

cells to chemotherapy

Acknowledgement

Dr. Jay Vadgama (Charles R. Drew University)

Drs. Susanne Henning, David Heber (UCLA Center for

Human Nutrition), Jonathan W. Said, Clara E. Magyar, Ngan

Doan (UCLA Department of Pathology)

Grant supports from the National Institutes of Health [NIH, NCI, NIMHD,

NCATS; Grant Nos. U54 CA143931-01, U54MD007598,

UL1TR000124 (J.V. Vadgama)], and NIH/National Center for Advancing

Translational Sciences UCLA CTSI [Grant No. KL2TR000122 (P.

Wang)]

enhanced chemopreventive effect by combining quercetin …...enhanced chemopreventive effect by...

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