CDK inhibitors as Targets of Chemoprevention...
Transcript of CDK inhibitors as Targets of Chemoprevention...
CDK inhibitors as Targets
of Chemopreventive Agents
Nam Deuk Kim, Ph.D.
Pusan National University
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Contents
Introduction
CDK Inhibitors, Cell Cycle and Cancer
Tamoxifen
Retinoids
Genistein
Resveratrol
Flavonoids
Indole-3-Carbinol
N-Acetylcycteine
Sulindac and Flavopiridol
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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.
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(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
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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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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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).
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RA causes a G1 arrest in OPM-2 cells
Fig. 6. Analyzed
by DNA flow
cytometry for
cell-cycle
distribution.
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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.
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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
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Fig. 12
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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
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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
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Resveratrol treatment resulted
in a concentration-dependent
induction of p53 and p21 at both
the transcriptional and
translational levels.
Fig. 20 30
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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).
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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.
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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
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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
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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.
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{(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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