Effects of metformin on Sirt1, Nrf2 and AhR expression in cancer cells

A Dissertation for the Degree of Doctor of Philosophy

Effects of metformin on Sirt1, Nrf2 and AhR

expression in cancer cells

Department of Pharmacy

Graduate School

Chungnam National University

By

Minh Truong Do

Advisor Hye Gwang Jeong

August 2014

Effects of metformin on Sirt1, Nrf2 and AhR expression

in cancer cells

Advisor Hye Gwang Jeong

Submitted to the Graduate School

in Partial Fulfillment of the Requirements

for the Degree of

Doctor of Philosophy

April, 2014

Department of Pharmacy

Graduate School

Chungnam National University

By

Minh Truong Do

i

Contents

Contents ---------------------------------------------------------------------------- i

List of Figures -------------------------------------------------------------------- vi

List of Abbreviations ---------------------------------------------------------- x

Abstract ----------------------------------------------------------------------------- 1

I. Introduction -------------------------------------------------------------------- 6

1. Metformin and reduced risk of cancer ------------------------------------- 6

2. Role of the AhR, CYP1A1 and CYP1B1 in carcinogenesis

and mechanisms of regulation of gene expression ---------------------- 6

3. Regulation of gene expression and role of Nrf2 and HO-1

in tumorigenesis and chemoresistance ------------------------------------ 9

4. Role of Sirt1 in tumorigenesis and chemoresistance --------------------- 11

II. Materials & Methods ------------------------------------------------------ 15

1. Materials ----------------------------------------------------------------------- 15

2. Cell culture and treatment --------------------------------------------------- 16

3. Measurement of cell viability and cytotoxicity --------------------------- 17

4. BrdU incorporation assay ---------------------------------------------------- 18

ii

5. RNA preparation and reverse transcription-polymerase

chain reaction (RT-PCR) ----------------------------------------------------- 19

6. Quantitative real-time RT-PCR (qRT-PCR) ------------------------------- 20

7. Luciferase and β-galactosidase assays ------------------------------------- 22

8. Western blotting --------------------------------------------------------------- 23

9. Preparation of nuclear and cytosolic extracts ----------------------------- 24

10. Immunoprecipitation (IP) ---------------------------------------------- 24

11. Chromatin immunoprecipitation (ChIP) ---------------------------------- 25

12. Small interfering RNA transfection --------------------------------------- 26

13. Sp1, HO-1, Sirt1, Pgc-1 and PPAR overexpression ----------------- 26

14. miR-34a inhibitor and mimic transfection ------------------------------- 27

15. Measurement of intracellular reactive oxygen species (ROS) -------- 27

16. Statistical analysis ----------------------------------------------------------- 28

III. Results ----------------------------------------------------------------------- 29

1. Metformin suppresses CYP1A1 and CYP1B1 expression in breast

cancer cells by down-regulating aryl hydrocarbon receptor

expression ------------------------------------------------------------------- 29

1.1. Metformin inhibits CYP1A1 and CYP1B1 expression

in breast cancer cells ------------------------------------------------------- 29

1.2. Down-regulation of AhR expression is required for the

iii

suppression of CYP1A1 and CYP1B1 by metformin ----------------- 33

1.3. Down-regulation of Sp1 by metformin inhibits AhR

transcriptional activity in breast cancer cells --------------------------- 38

1.4. Inhibition of CYP1A1 and CYP1B1 expression by

metformin is independent of ER ---------------------------------------- 41

1.5. Metformin suppresses endogenous AhR-ligand-induced

CYP1A1 and CYP1B1 expression by reducing TDO

expression in breast cancer cells ------------------------------------------ 43

1.6. Metformin suppresses TDO expression by down-regulating

the Sp1/glucocorticoid receptor (GR) signaling pathway ------------- 47

2. Metformin inhibits heme oxygenase-1 expression in cancer cells

through inactivation of Raf/ERK/Nrf2 signaling

and AMPK-independent pathways --------------------------------------- 52

2.1. Metformin suppresses HO-1 expression in cancer cells --------------- 52

2.2. Metformin suppresses Nrf2 expression through a Keap1-

independent mechanism in cancer cells --------------------------------- 54

2.3. Metformin suppresses Nrf2 expression in cancer cells via

Raf-ERK inactivation ------------------------------------------------------ 58

2.4. Down-regulation of HO-1 expression by metformin

is independent of AMPK -------------------------------------------------- 61

iv

2.5. Reduction of HO-1 contributes to anti-proliferative effects

of metformin in cancer cells ---------------------------------------------- 65

3. Metformin induces microRNA-34a to down-regulate Sirt1/Pgc-1/Nrf2

pathway leading to increased susceptibility of cancer cells to

oxidative stress and therapeutic agents ---------------------------------- 71

3.1. Metformin suppresses Sirt1 expression in p53 wild-type

cancer cells ------------------------------------------------------------------ 71

3.2. p53-dependent induction of miR-34a is required for the

reduction of Sirt1 by metformin ------------------------------------------ 73

3.3. Down-regulation of Sirt1 by metformin inhibits Nrf2 expression

and increases susceptibility of wild-type p53 cancer cells

to oxidative stress ----------------------------------------------------------- 77

3.4. Metformin inhibits Nrf2 expression mediated by

suppression of Pgc-1 ----------------------------------------------------- 83

3.5. Metformin suppresses Nrf2 expression by inhibiting

PPAR transcriptional activity and attenuating PPAR

binding to the Nrf2 promoter --------------------------------------------- 86

3.6. Up-regulation of DR5 expression by metformin sensitizes

wild-type p53 cancer cells to TRAIL-induced apoptosis ------------- 90

v

IV. Discussion ----------------------------------------------------------------- 97

V. Conclusion ------------------------------------------------------------------ 118

VI. References ----------------------------------------------------------------- 120

Abstract in Korean ----------------------------------------------------------- 147

Appendix ------------------------------------------------------------------------ 150

vi

List of Figures

1. Metformin suppresses CYP1A1 and CYP1B1 expression in breast

cancer cells by down-regulating aryl hydrocarbon

receptor expression

Fig. 1. Metformin down-regulates CYP1A1 and CYP1B1 transcription

in MCF-7 breast cancer cells ------------------------------------------ 31

Fig. 2. Metformin down-regulates AhR expression in MCF-7

breast cancer cells ------------------------------------------------------ 35

Fig. 3. Down-regulation of AhR expression is required for the reduction

of CYP1A1 and CYP1B1 by metformin in MCF-7 cells ---------- 37

Fig. 4. The reduction in Sp1 protein levels mediated by metformin

suppresses AhR transcriptional activity in MCF-7

breast cancer cells ------------------------------------------------------- 39

Fig. 5. Metformin down-regulates CYP1A1 and CYP1B1 expression

in ER-negative MDA-MB-231 breast cancer cells --------------- 42

Fig. 6. Metformin attenuates endogenous AhR ligand-induced CYP1A1

and CYP1B1 expression by reducing tryptophan-2,3-dioxygenase

expression in MCF-7 breast cancer cells ----------------------------- 45

Fig. 7. The down-regulation of TDO expression by metformin

is mediated via down-regulation of Sp1 and GR proteins --------- 49

vii

Fig. 8. Proposed signaling pathways underlying the effects of

metformin on down-regulation of CYP1A1 and

CYP1B1 expression in breast cancer cells ------------------------- 51

2. Metformin inhibits heme oxygenase-1 expression in cancer cells

through inactivation of Raf/ERK/Nrf2 signaling and

AMPK-independent pathways

Fig. 9. Metformin down-regulates HO-1 expression

in various cancer cells ------------------------------------------------- 53

Fig. 10. Effects of metformin on Nrf2 and Keap1 expression

in cancer cells ----------------------------------------------------------- 56

Fig. 11. Inactivation of Raf-ERK signaling by metformin is required

for down-regulation of Nrf2 expression in cancer cells ---------- 59

Fig. 12. Metformin suppresses HO-1 expression in cancer cells

in an AMPK-independent manner ----------------------------------- 63

Fig. 13. Effects of metformin on proliferation of cancer cells ------------- 67

Fig. 14. Role of HO-1 suppression in anti-proliferative effect

of metformin in cancer cells ------------------------------------------ 69

Fig. 15. Proposed signaling pathways underlying the effects of metformin

on down-regulation of HO-1 expression in cancer cells ---------- 70

viii

3. Metformin induces microRNA-34a to down-regulate Sirt1/Pgc-1/Nrf2

pathway leading to increased susceptibility of cancer cells to

oxidative stress and therapeutic agents

Fig. 16. Metformin down-regulates Sirt1 expression in wild-type p53

cancer cells -------------------------------------------------------------- 72

Fig. 17. Metformin increases induction of p53 protein and miR-34a

in wild-type p53 cancer cells ----------------------------------------- 75

Fig. 18. p53-dependent induction of miR-34a is required for Sirt1

reduction by metformin ----------------------------------------------- 76

Fig. 19. Metformin-mediated down-regulation of Sirt1 inhibits Nrf2

expression in MCF-7 breast cancer cells ----------------------------- 79

Fig. 20. Effects of metformin on intracellular ROS production,

H2O2-induced cytotoxicity and apoptosis --------------------------- 81

Fig. 21. Metformin inhibited Nrf2 expression is mediated

by Pgc-1 suppression ------------------------------------------------ 84

Fig. 22. Metformin inhibits Nrf2 expression mediated by suppressing

transcriptional activity of PPAR ------------------------------------- 88

Fig. 23. Effects of metformin on CHOP and DR5 expression

in breast cancer cells ---------------------------------------------------- 92

Fig. 24. Metformin sensitizes wild-type p53 cancer cells to

TRAIL-induced apoptosis --------------------------------------------- 93

ix

Fig. 25. Proposed signaling pathways underlying the effects of

metformin on Sirt1/Pgc-1/Nrf2 pathway leads to increased

susceptibility of cancer cells to oxidative stress and

therapeutic agents ---------------------------------------------------- 96

x

List of Abbreviations

3’-UTR 3’-Untranslated region

4-OHE2 4-Hydroxyestradiol

AhR Aryl hydrocarbon receptor

AMPK AMP-activated protein kinase

ARE Antioxidant response element

ARNT Aryl hydrocarbon receptor nuclear translocator

BrdU 5-Bromo-2’-deoxyuridine

ChIP Chromatin immunoprecipitation

CHOP C/EBP homology protein

CO Carbon monoxide

CRE Cyclic AMP response element

CYP Cytochrome P450

DMEM Dulbecco's modification of Eagle's medium

DN-AMPK Dominant-negative form of AMPK

DR5 Death receptor 5

ECL Enhanced chemiluminescence

ELISA Enzyme-linked immunosorbent assay

ER Estrogen receptor

ERK Extracellular signal-regulated kinase

xi

FOXO Forkhead homeobox type O

GAPDH Glyceraldehyde 3-phosphate dehydrogenase

GR Glucocorticoid receptor

HER2 Human epidermal growth factor receptor 2

HO-1 Heme oxygenase-1

Hsp90 Heat shock protein 90

IgG Immunoglobulin G

IP Immunoprecipitation

Keap1 Kelch-like ECH-associated protein 1

LDH Lactate dehydrogenase

MAPK Mitogen-activated protein kinase

miR-34a MicroRNA-34a

MTT 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium

bromide

NF-B Nuclear Factor kappa B

Nrf2 Nuclear erythroid factor 2 (NE-F2)-related factor 2

PARP Poly-ADPribose polymerase

Pgc-1 Peroxisome proliferator-activated receptor gamma

coactivator-1 alpha

PPAR Peroxisome proliferator-activated receptor gamma

xii

PPRE PPAR-responsive elements

qRT-PCR Quantitative real-time RT-PCR

RNU6B U6B small nuclear RNA

ROS Reactive oxygen species

RT-PCR Reverse transcription-polymerase chain reaction

SDS-PAGE Sodium dodecyl sulphate polyacrylamide gel

electrophoresis

siRNA Small interfering RNA

Sirt1 Sirtuin1

SOD2 Superoxide dismutase 2

Sp1 Specificity protein 1

TCDD 2,3,7,8-Tetrachlorodibenzo-p-dioxin

tBHQ tert-Butylhydroquinone

TDO Tryptophan-2,3-dioxygenase

TNF Tumor necrosis factor

TRAIL Tumor necrosis factor–related apoptosis-inducing ligand

XRE Xenobiotic responsive elements