CDK inhibitors as Targets

of Chemopreventive Agents

Nam Deuk Kim, Ph.D.

Pusan National University

1

Contents

Introduction

CDK Inhibitors, Cell Cycle and Cancer

Tamoxifen

Retinoids

Genistein

Resveratrol

Flavonoids

Indole-3-Carbinol

N-Acetylcycteine

Sulindac and Flavopiridol

2

1. Introduction Chemoprevention is a promising approach in the use of

natural or synthetic compounds to inhibit neoplastic

development.

Delaying the onset of cancer for 15-20 years may

significantly increase the standard of living for an

individual.

For the purposes of this chapter, we are going to focus

on agents that target components involved in cell

proliferation and differentiation.

3

(오렌지껍질)

Curry(카레)

녹차 Red raspberry(복분자)

Resveratrol, 포도

Garlic, 마늘 Broccoli sprouts (브로콜리)

대표적 화학적암예방제

복분자 녹차

포도

카레 브로콜리

마늘

오 렌 지

• 폴리페놀 • 라이코펜 • 캡사이신 • 플라보노이드

석류 추출물에 의한 인체 유방암의 화학적 암 예방 및 보조 치료제 가능성 (2002)

IF = 4.431

Phytoestrogens

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2. CDK Inhibitors, cell cycle and cancer

Fig. 1. Potential cell targets of various chemopreventive agents. Tamoxifen (Tam),

Retinoic acid (RA), Indole-3-Carbinol (I3C), and Apigenin appear to target pRb

protein phosphorylation. Resveratrol and N-acetyl-cysteine (NAC) appear to induce

a G1 arrest and to target p21. NAC also targets p16. Resveratrol also induces a G2

arrest. Genistein appear to induce a G2 arrest and affect p21 expression.

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10

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3. Tamoxifen

Tamoxifen (Tam, (Z)-2-[4-(1,2-diphenylbut-1-enyl)phenoxy]-

N,N-dimethylethanamine): used for the prevention of breast

cancer, as well as other types of cancer.

Tam: a nonsteroidal antiestrogen, a competitive inhibitor of

estradiol.

Originally synthesized as an antifertility drug.

Low conc. of Tam – cytostatic effect against breast ca.

- arrested in early G1

Higher conc. of Tam – cytotoxic effect against breast ca.

- cell death

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Fig. 3. Tam caused a decrease in Rb

protein level. By “decrease in CDK2

activity”.

Fig. 2. Analysis of [3H]thymidine

incorporation revealed that tamoxifen

caused a steady decrease in DNA

synthesis over the time course with a

60% inhibition after 96 h of treatment.

Effects of I3C and tamoxifen on in vitro CDK2 kinase activity in MCF-

7 cells. MCF-7 cells were treated with I3C and/or tamoxifen (Tam) for

48 h. CDK2 was immunoprecipitated from cell lysates and assayed

for in vitro kinase activity using the COOH terminus of the Rb protein

as a substrate (GST-Rb).

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Switching from Tamoxifen to Aromatase inhibitor

improves survival in early breast cancer

Drugs known as aromatase inhibitors have produced

superior results to tamoxifen alone in the treatment of

ER-positive, postmenopausal breast cancer.

Arimidex(anastrozole), Aromasin(exemestane), and

Femara(letrozole)

In clinical trials, it was apparent that patients who

switched to an aromatase agent had a 39% reduced risk

of death compared to those who continued treatment

with tamoxifen. Reference : Boccardo F, Rubagotti A, Aldrighetti D, et al. Switching to an aromatase inhibitor provides mortality benefit in early breast

carcinoma. Cancer. 2007; 109: 1060-1067

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15

H OH

O

Androstenedione

Estrone Formic acid

O2

2NADPH/H+

H2O 2NADP O2

2NADPH/H+

H2O 2NADP

H2O Fe3+OH2

2NADP

+

Fe3+OOH O2

2NADPH/H+

enolization

O

O O

HO

O

HO

HO

O

HO

HOOH

O

HO

O

O

HO

OOHO

Fe

O

HO

Aromatization of Androstenedione to Estrone

Aromatase

Cytochrome P450 Superfamily

Aromatization Catalyst of Androgen to Estrogen by 3 Oxidation Successively

CYP19 Gene

Rate-Limiting Step in Estrogen Production

4. Retinoids

Retinoic Acid (RA) is a metabolite of vitamin A.

All-trans-retinoic acid: (2E,4E,6E,8E)-3,7-dimethyl-9-

(2,6,6-trimethylcyclohexen-1-yl)nona-2,4,6,8-tetraenoic

acid

It has been known for 75 yrs that Vit. A deficiency in the

rat model causes distinct effects on both cell proliferation

and differentiation.

Treatment of human myeloma cell lines with trans RA

down regulated expression of interleukin-6 (IL-6)

receptor expression, which is a major growth factor

receptor in OPM-2 human myeloma cells.

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Fig. 4. All-trans RA (ATRA)

caused

dephosphorylation of Rb

protein in OPM-2 cells.

Fig. 5. Expression of

p21 increased after a

24 h exposure to all-

trans RA (ATRA).

17

RA causes a G1 arrest in OPM-2 cells

Fig. 6. Analyzed

by DNA flow

cytometry for

cell-cycle

distribution.

18

The acyclic retinoid (ACR)-mediated G1 arrest is associated with

increase in protein levels of p21CIP1 and suppress cyclin D.

Fig. 7

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Fig. 8. ACR causes an

increase in the cellular

level of both the RARβ

protein and mRNA,

Time-dependent effects of ACR on the

expression of RARβ and cell cycle control

molecules.

Fig. 9

Fig. 10

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5. Genistein

One of several known isoflavones.

Isoflavones, such as genistein and daidzein, are found in a number of plants including lupin, fava beans, soybeans, kudzu, and psoralea being the primary food source, also in the medicinal plant, Flemingia vestita and coffee.[

Cultures with diets rich in soy products have a lower incidence/mortality rate in certain types of cancer.

Genistein decreased the formation of 7,12-DMBA induced mammary tumors.

Chemopreventive activity in prostate cancer.

Hormone-independent effects, such as inhibition of protein tyrosine kinase activity, DNA topoisomeraseⅡ activity, ROS formation.

21

5,7-Dihydroxy-3-(4-hydroxyphenyl)chromen-4-one

Genistein causes a G2/M arrest in both ER positive

and ER negative breast cancer cell lines.

Fig. 11

22

Fig. 12

23

Inhibition in cell cycle associated with the

induction of p21 expression.

Fig. 13 Fig. 14

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6. Resveratrol

Resveratrol (3,5,4’-trihydroxystilbene) occurs naturally in

a variety of plants including grapes, peanuts.

Come from “red wine”.

Resveratrol may be protective against oxidation of

lipoproteins, inhibit platelet aggregation and alter

eicosanoid synthesis.

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Fig. 15. The structure of resveratrol

is similar to the synthetic estrogen

diethylbestrol.

Fig. 16. In the absence of

estrogen, resveratrol behaves as

a partial agonist.

In the presence of estrogen,

resveratrol behaves as an

antagonist.

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Antiproliferative effect of Resveratrol in

Human Prostate Carcinoma

Growth inhibition by resveratrol

Fig. 17

27

Morphological changes and apoptotic cell death induced by

resveratrol.

Fig. 18

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Effect of resveratrol on levels of cell cycle regulators.

Decrease in the protein expression of D-type cyclins and

Cdk4, but did not affect the protein expression of cyclin A,

cyclin E, Cdk2, and Cdc2.

Fig. 19

29

Resveratrol treatment resulted

in a concentration-dependent

induction of p53 and p21 at both

the transcriptional and

translational levels.

Fig. 20 30

31

7. Flavonoids

Flavonoids naturally occur in plants and vegetables and

have been shown to have chemopreventive benefits in

laboratory models.

Grape polyphenols, apigenin and epigallocatechin-3-

gallate(ECGC).

Flavonoids in red wine : catechin, epicatechin and

quercetin.

Grape seed polyphenolic fraction (GSP).

32

Anticarcinogenic effect of grape seed polyphenolic

fraction (GSP) in human prostate carcinoma

DU145 cells.

Fig. 21. GSP inhibited the growth of

DU-145 cells and caused their death in

both a time- and dose- dependent

manner.

33

Treatment with GSP resulted in a

significant induction of p21, a

decrease in CDK4 protein expression

and G1 cell cycle arrest.

Fig. 22 Fig. 23

Fig. 24 34

Green and black tea are another source for

flavonoids

Tea contains the polyphenol epigallocatechin-3-

gallate(EGCG) present in the leaves and stems of tea

plant.

Fig. 26. Effects of EGCG on growth of MCF-7

cells and cell cycle progression.

Fig. 25. Treatment of EGCG induced a G1 arrest.

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Mechanisms of growth arrest by ECGC in

MCF-7 cells

• Hypophosphorylation of pRb.

• Inhibition of CDK2 and CDK4 kinase activities.

• Induction of p21 and p27.

Fig. 27

Fig. 28

Fig. 29 36

Inhibition of HIF-1 alpha and VEGF expression by apigenin

Molecular Carcinogenesis 2008 47(9):686-700.

1. HIF-1α expression 2. VEGF expression

Apigenin is another naturally occurring

flavonoid found in fruits and vegetables

Mechanism of the apigenin – induced G1 arrest in

human diploid fibroblasts (HDF).

Fig. 30. Western blot analysis of apigenin-

treated HDF indicated that cyclin D1 was

expressed at higher levels than in untreated

cells, which signifies that they were arrested

in G1 phase rather than in a G0 quiescent

state.

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5,7-Dihydroxy-2-(4-hydroxyphenyl)-4H-1-

benzopyran-4-one

• The G1arrest was further studied by cyclin-dependent kinase 2 (cdk2)

immune complex–kinase assays of apigenin treated HDF, which

demonstrated a dose-dependent inhibition of cdk2 by apigenin.

• Inhibition of cdk2 kinase activity in apigenin-treated cells was

associated with the accumulation of the hypophosphorylated form (105-

kDa) of the retinoblastoma (Rb) protein as measured by Western blot

analysis.

Fig. 31 Fig. 32

39

40

Apigenin-induced Apoptosis is Enhanced by Inhibition of

Autophagy Formation in Human Colon Cancer Cells

2013.12.03

Lab. of Cancer Biology

Yu Jin Lee, B.S.

Supervised by Nam Deuk Kim, Ph.D.

Indole–3-Carbinol

Indole-3-carbinol (I3C, 1H-indol-3-

ylmethanol) is a compound found in high

concentrations in Brassica family

vegetables, including broccoli, cauliflower,

Brussels sprouts, and cabbage.

As a nutritional supplement, I3C has received attention

in recent years as a promising preventive and treatment agent for breast

and other types of cancers, and may have

beneficial effect in the management of Herpes simplex virus (HSV) and human papilloma virus (HPV).

Fig. 33

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I3C inhibit cell cycle progression at the G1 checkpoint and

elevate p53 tumor suppression levels

p53 is required for the I3C-induced

arrest of MCF10A cells

Fig. 34

Fig. 35

42

Phosphorylation of p53 at the N-

terminus has been shown to disrupt

the interaction between p53 and its

ubiquitin ligase, MDM2, and

therefore stabilizing p53.

I3C stabilizes phosphorylated forms of p53

Phosphorylation of p53 at several

N-terminal residues represents a

key regulatory mechanism that

disrupts the p53–MDM2

interaction in response to DNA

damage.

Proposed model of I3C induced G1

cell cycle arrest by

activation of the phosphorylation of

p53.

Fig. 36 43

Only I3C treatment caused the stable production of p53 phosphorylated at serines 6,

9, 15 and 392 at all time points tested.

The time course of I3C induced p53 phosphorylation correlated with the I3C

stimulation of p21 production.

Therefore, the level of

phosphorylated p53 was

examined in indole-

treated and untreated

cells by Western blotting

over a 72 hr time course

Fig. 37

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9. N-Acetylcysteine

N-acetylcysteine (NAC) is a small molecule that is known

to possess antitumor activity in organs such as skin,

lung, liver and colon.

NAC is clinically used for the treatment of chronic

bronchitis and paracetamol poisoning.

Treatment of 308 papilloma cells with NAC prolonged

cell cycle progression through G1 and induced p16 and

p21.

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10. Sulindac and Flavopiridol Sulindac is a NSAID that has been shown to inhibit both

COX-1 and COX-2.

Although the exact mechanism of chemoprevention is

unclear, it has been shown that sulindac and derivatives

thereof can inhibit growth and induce apoptosis in cancer

models.

Flavopiridol is a flavone derivative that can induce cell

cycle arrest and apoptosis in numerous transformed cell

lines.

46

{(1Z)-5-fluoro-2-methyl-1-[4-(methylsulfinyl)benzylidene]-1H-

indene-3-yl}acetic acid

2-(2-chlorophenyl)-5,7-

dihydroxy-8-[(3S,4R)-

3-hydroxy-1-methyl-4-

piperidinyl]-4-

chromenone

It has been shown to reduce CDK2 and CDK4 kinase

activity in human breast carcinoma cell lines.

Downregulate cyclinD1 in esophageal and breast

carcinoma cell lines.

Induce apoptosis in non-small cell lung cancer and

esophageal cancer cell lines.

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Conclusions Affect cell cycle, cell death and differentiation

pathways.

Many of these chemopreventive agents have

pleiotropic effects within the cell.

Continued studies of the cellular effects and

molecular mechanism will lead to a greater

understanding and increase the promise of

chemopreventive therapy.

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1. Text 2, 3 Carolyn M. Cover, S, et al. Indole-3-Carbinol and Tamoxifen Cooperate to Arrest the Cell Cycle of MCF-7 Human Breast Cancer Cells. [CANCER RESEARCH 59, 1244–1251, March 15, 1999] 4, 5, 6 By Yi-Hsiang Chen, Donald Lavelle, et al. Growth Inhibition of a Human Myeloma Cell Line by All-trans Retinoic Acid Is Not Mediated Through Downregulation of Interleukin-6 Receptors but Through Upregulation of p21WAF1. Blood, Vol 94, No 1 (July 1), 1999: pp 251-259 7, 8, 9, 10 Masahito Shimizu,1 Masumi Suzui, et al. Effects of Acyclic Retinoid on Growth, Cell Cycle Control, Epidermal Growth Factor Receptor Signaling, and Gene Expression in Human Squamous Cell Carcinoma Cells. Clinical Cancer Research Vol. 10, 1130–1140, February 1, 2004 11, 12, 13, 14 Zhi-Ming Shao, Mary L. Alpaugh , et al. Genistein Inhibits Proliferation Similarly in Estrogen Receptor-Positive and Negative Human Breast Carcinoma Cell Lines Characterized by P21WAF1/CIP1 Induction, G2/M Arrest, and Apoptosis. Journal of Cellular Biochemistry 69:44–54 (1998) 15. Eun-Jung Park and Sang Kook Lee. Anti-inflammatory Effects of Resveratrol and Its Analogs: COX-2 and iNOS as Potential Targets. Cancer Prevention Research Vol. 10, No. 2, 2005

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